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https://f1000research.com/articles/9-347/v1
|
11 May 20
|
{
"type": "Research Article",
"title": "Workplace violence against security personnel at a university hospital in Egypt: a cross-sectional study",
"authors": [
"Ahmed A. Albadry",
"Abdel-Hady El-Gilany",
"Hala Samir Abou-ElWafa",
"Abdel-Hady El-Gilany",
"Hala Samir Abou-ElWafa"
],
"abstract": "Background: Violence is common among security personnel. To the best of the authors' knowledge no recent studies have investigated this problem. This study aimed to estimate the prevalence and associated factors of violence against hospital security personnel and describe circumstances of violence, type of perpetrators, and victims’ response. Methods: In total, 170 security personnel from a university hospital in Egypt were recruited in this cross-sectional study. Data were collected using the Arabic version of a questionnaire developed by the International Labour Office. Results: The majority (87.3%) of security personnel reported violence exposure in the past year. Being a woman and working more than 5 years were independent predictors of violence exposure. The commonest forms of physical violence were pushing and beating. Verbal abuse and threats were the commonest emotional violence. Patients and their relatives/friends were the commonest perpetrators of violence. Conclusions: Violence is common among hospital security personnel in this setting. Adequate training and recruitment of more security personnel may contribute to decreasing violence.",
"keywords": [
"Hospital violence",
"Security personnel",
"occupational exposure"
],
"content": "Introduction\n\nWorkplace violence (WPV) is well-defined as any act or danger of physical violence, harassment, bullying, or other disruptive activities at the workplace that may result in physical or emotional problems1. The literature and news show that violence is common globally and occurs every day in various public spheres, including the health system2.\n\nHealthcare violence reflects violence in society in general. However, violence contradictory to social expectations that hospitals are sites that denote security, care, sympathy, and lifesaving. It is hard to acknowledge that violent incidents occur commonly in hospitals and in some situations physical violence in hospitals can be exceedingly dangerous3.\n\nHealthcare WPV is an underestimated and constant problem that has been widely overlooked. Many healthcare organizations and institutions that are considered safe resorts are now facing “steadily growing rates of crime, including violent criminalities such as stabbing, rape, and homicide” as stated by Phillips4. According to the US Agency of Labor Statistics 2007 report, violence occurs more habitually in healthcare and social support organizations than in any other labor force segment and is responsible for 60% of all nonfatal assaults within this segment5.\n\nThe majority of research studies have revealed that following an incident of WPV, there are high rates of lost working days, burnout, and dissatisfaction, in addition to decreased feelings of wellbeing among staff members6,7. In return, fright at work has even driven some healthcare personnel to protect themselves by carrying weapons, mostly firearms or knives8.\n\nHospital security officers are required to protect the safety of healthcare workers, hospital guests, and patients. They are asked to assist in control of violent situations and are consequently at an elevated risk to tolerate violence-associated incidences and are therefore recognized as a group at high risk of being attacked9. They are often disregarded in the literature concerning healthcare violence-related incidences. The comparatively few studies that do involve hospital security staff demonstrate that these workers are among groups with the highest rates of violence-related incidences within the healthcare setting10–12.\n\nThe significance of policies and security staff training has been accepted and various hospitals offer limitation tools and weapons to security personnel. Such establishment, chiefly of non-lethal intermediary weapons (e.g. conducted electrical weapons) or lethal weapons (e.g. handguns) has been conflicted owing to the moral, legal, and financial issues related to the well-being and safety of patients and staff in hospitals13,14.\n\nWPV directed towards hospital security staff has seldom been studied in developing countries including Egypt; thus, the actual magnitude of the problem is unknown. The purposes of this study are to estimate the prevalence and associated issues of different types of violence against hospital security personnel, conditions of violence, category of perpetrators, and victims’ reaction in a university hospital setting.\n\n\nMethods\n\nThis descriptive cross-sectional study was conducted among formal security personnel at a university hospital from September 1, 2018 to October 31, 2018.\n\nAll formal security personnel were the target population. The inclusion criteria were permanent or temporary workers employed for 1 year or more.\n\nApproval for the study was obtained from the Institutional Research Board at the university hospital (proposal number: R.18.07.236). The name of the university hospital has been blinded to protect anonymity of participants. Written informed consent of study participants to participate willingly in the study with the right to withdraw from the research at any time was obtained with a guarantee of confidentiality and anonymity of the information.\n\nWorkers fulfilling the eligibility criteria were interviewed at their workplace at the beginning of the workday before their work shift after arrangement with their direct supervisor in the supervisor room. The English version of the questionnaire developed by the International Labor Office, International Council of Nurses, World Health Organization, and Public Services International (2003) regarding WPV in the health sector was used in Arabic (English version available here: https://www.who.int/violence_injury_prevention/violence/interpersonal/en/WVquestionnaire.pdf. A modified Arabic version of this questionnaire was developed and tested for validity and reliability in a previous study in Saudi Arabia15. The questionnaire was used to collect the following information: demographics and workplace characteristics; WPV in the past year; its nature, frequency, response, consequences, and satisfaction of incident handling; policies, and strategies to deal with the incident.\n\nData were statistically analysed using the Statistical Package for Social Sciences (SPSS) version 20. Data were presented in the form of numbers and %. Chi-squared test was used to determine significant risk factors of violence. Multivariate logistic regression analysis using forward Wald method was run to identify significant independent predictors of violence. Crude and adjusted odds ratios and their 95% confidence intervals were considered. P≤0.05 was considered statistically significant.\n\n\nResults\n\nThe questionnaire was completed by 150 out of 170 legible security personnel (response rate 88.2%) who reported a total number of 553 workplace events in the past year.\n\nThe majority (87.3%) of security personnel reported exposure to violence in the past year. Being a woman and working for over 5 years were independent predictors of violence exposure (adjusted odds ratio (AOR) = 7.7 and 10.1, respectively; Table 1).\n\nr=reference group. **P≤0.01 ***P≤0.001.\n\nTable 2 shows that the commonest suggestions from participants to prevent violence were training (73.3%) and assigning a large number of personnel (62.7%), while Table 3 shows that the commonest forms of physical violence reported by personnel were pushing (24.8%), beating (18.6%) and throwing objects (12.3%). The threat of physical force and verbal abuse were the commonest emotional violence experienced by participants (23.7% and 20.4%; respectively). Patients’ relatives/friends and patients themselves were the commonest perpetrators of violent events (72.5% and 23.3%; respectively).\n\nTable 4 shows that security personnel were alone in more than one-third of WPV events. In addition, more than two-fifths and more than one-third of violent events occurred during evening shifts and official vacation other than Friday, respectively. More than one-fifth of violent events were reported to the hospital administration. Only 3.8% of perpetrators received a verbal warning from the directors and only 2% of 553 reported events resulted in a lawsuit against the perpetrator.\n\nTable 5 shows that participants reported that the commonest effect of violence was being bothered (80.9%), being fearful (69.5%), and having work dissatisfaction (61.8%) and anger/anxiety (47.3%). The commonest coping mechanisms were reporting to directors/supervisors (94.7%), telling family/friends (92.4%), pretending the event did not occur (77.1%) and replying to perpetrators themselves at the time of the event (74.0%).\n\nCategories are not mutually exclusive.\n\n\nDiscussion\n\nHospital security workers, who are trained to offer tertiary prevention on escalation of an event, have also been acknowledged as a group at an increased risk of being assaulted12,16 and in need of tools to identify, alleviate, and avoid violent events in hospitals17.\n\nIn this study, the majority (87.3%) of security personnel reported exposure to violence in the past year. A similarly high prevalence (63.8%) of type II violence, which is perpetrated by a client receiving services from an organization, such as a patient or guest, was reported among security guards and police officers in a study of six US hospitals16. This high prevalence could be attributed to the nature of the work of security personnel, who repeatedly interfere in efforts to protect both staff and patient safety and are frequently injured in occurrences with violent patients18. It has been previously acknowledged that hospital safety and security employees are often ignored in the healthcare violence-related injury literature9. There are relatively few studies involving hospital security workers and these have revealed that these employees have some of the highest rates of violence-related incidences within hospitals, anywhere from 2 to 5 times as many incidences as nurses10–12,19–21. However, almost no studies have been carried out to discuss the risk and protective factors for hospital security employees9.\n\nIn this study, being a woman and working for more than 5 years are independent predictors of violence exposure (AOR=7.7 and 10.1; respectively). Several studies reported that younger and less experienced security officers have elevated rates of violence-related incidences8,12,22–25.\n\nIn contrast to these findings, a study conducted in the Midwest US, showed that both age and gender were not associated with verbal violence, although, while healthcare workers more than 60 years old were less exposed to physical violence (odds ratio (OR) = 0.31, p<0.05)26. In another study of US hospitals, the higher prevalence of WPV across all sub-types in workers of a younger age suggested that younger workers are more prone to be victims16. Older workers may be more tolerant of these events resulting in their under reporting27 or they may be more skilful at event de-escalation.\n\nMost studies that investigated gender of recipients of violence-related incidences in healthcare reported that men are more liable to experience these injuries more than women11,12,23,28–30, while others have not found difference in rates based on victims’ gender31,32.\n\nThe commonest suggestions to prevent violence in the current study were training (73.3%) and assigning a larger number of personnel (62.7%). The results of a survey of healthcare specialists from 19 hospitals in six cities of Heilongjiang Province, China are in agreement with our results, where the respondents exposed to WPV expected to obtain organizational and social support. Those exposed to psychological violence had a strong opinion of the need for targeted training to support their proficiency in responding to violence (OR = 1.319, 95% CI: 1.034–1.658) and endorsing WPV legislation (OR = 1.968, 95% CI: 1.523–2.543). Those exposed to physical violence incidences thought it might be helpful to strengthen staff with back-up support (OR = 3.101, 95% CI: 1.085–8.860)33.\n\nThe commonest forms of physical violence in this study were pushing (24.8%), beating (18.6%) and throwing objects (12.3%). The threat of physical force and verbal abuse were the commonest emotional violence experienced (23.7% and 20.4%; respectively). Patients’ relatives/friends and patients themselves were the commonest perpetrators of violent events (72.5% and 23.3%; respectively). Similarly, verbal abuse (32.8%) was the most prevalent subtype of type II violence among security guards and police officers in the US followed by physical threat (24.1%), then finally physical assault (6.9%)16.\n\nAlso, in the Emergency Department (ED) in Ankara, Turkey, regarding frequency of physical violence, security officers (75%) described higher rates than other professions working in the ED (p<0.0001); exposure to any form of verbal abuse was highest among housekeepers (90.9%) and security officers (90.6%). In that study, self-reported verbal threat and sexual harassment among security officers was 75% and 15.6%, respectively. Relatives or friends accompanying patients were most frequently responsible for the violence, and this confirms what was concluded in a previous study34.\n\nIn a study within a large hospital system in Midwest US, nurses (OR = 1.87, p≤0.01) and security personnel (OR = 4.71, p≤0.01) reported verbal violent events in the past year more than any other job groups. Also, security staff (OR = 30.79, p≤0.001), registered nurses (OR = 2.72, p≤0.05), and mental health specialists (OR = 18.71, p≤0.01) were at a higher risk for physical violence26. These findings concerning physical violence consolidate other previous studies that have documented security personnel, mental health specialists, and nurses as being at higher risk11,16.\n\nIn this study, security personnel were alone in more than one-third of events. Additionally, more than two-fifths and more than one-third of violent events occurred during evening shifts and official vacation other than Friday, respectively. More than one-fifth of violent events were reported to the hospital administration. Only 3.8% of perpetrator received a verbal warning from the directors and only 2% had a lawsuit against the perpetrator. In a study from Israel, security employees emphasized the contribution of the behaviour of physicians and nurses to the development of a violent incident, especially verbal interaction, and delayed response time. A previous study reported that clinical staff had a negative attitude towards security personnel and considered that calling them to situations is a contributing factor to the development of a violent episode, which can escalate the situation and should be avoided if not necessary2.\n\nIn the present study, the commonest effects of violence were being bothered (80.9%), being fearful (69.5%), and having work dissatisfaction (61.8%) and anger/anxiety (47.3%). The commonest coping mechanisms were reporting to directors/supervisors (94.7%), telling family/friends (92.4%), pretending the event did not occur (77.1%) and replying to perpetrators (74.0%). Similarly, the most commonly reported reaction of ED staff in Turkey was sadness for sexual harassment (86%), physical assault (82.9%) and verbal threats (82%). The other reactions were anger for physical assault (83.8%), disappointment for verbal threat (79.3%) and disgust for physical assaults (69.3%). However, the most common coping method was “Doing nothing and keeping silent” (37.2–59.5%), followed by reporting to a manager (56.8%) for physical assaults, but this method is the least commonly used among participants experiencing verbal abuse (35.8%), verbal threat (34.7%) and sexual harassment (34.9%)34.\n\nAs this is a small-scale study in a single hospital, its results cannot be generalized to all health facilities. The possibility of overestimating the problem by security personnel to gain sympathy or more incentives cannot be excluded.\n\nFrom the results of this study, we recommend that security personnel at university hospitals need more training, and more adequate numbers should be recruited to cover shifts and time points with high risk of violence. The feasibility of other countermeasures suggested by security personnel need to be tested in further intervention studies before being adopted. Routine notification, analysis and record keeping of WPV events should be mandatory to monitor changes in the magnitude of the problem.\n\n\nData availability\n\nHarvard Dataverse: Workplace violence against security personnel at a university hospital in Egypt, https://doi.org/10.7910/DVN/FRVSR235.\n\nThis project contains the following underlying data:\n\n- Datasheet containing all variables obtained from the questionnaire for all participants.\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\nStene J, Larson E, Levy M, et al.: Workplace violence in the emergency department: giving staff the tools and support to report. Perm J. 2015; 19(2): e113–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShafran-Tikva S, Chinitz D, Stern Z, et al.: Violence against physicians and nurses in a hospital: How does it happen? A mixed-methods study. Isr J Health Policy Res. 2017; 6(1): 59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelloway EK, Barling JH editors: In: Handbook of workplace violence. 1st ed. Sage Publications. 2006; 47–68. Reference Source\n\nPhillips JP: Workplace Violence against Health Care Workers in the United States. N Engl J Med. 2016; 374(17): 1661–9. PubMed Abstract | Publisher Full Text\n\nJanocha JA, Smith RT: Workplace Safety and Health in the Health Care and Social Assistance Industry, 2003-07. US Bur Labor Stat. 2010; (2): 1–20. Reference Source\n\nTaylor JL, Rew L: A systematic review of the literature: workplace violence in the emergency department. J Clin Nurs. 2011; 20(7–8): 1072–85. PubMed Abstract | Publisher Full Text\n\nHanson GC, Perrin NA, Moss H, et al.: Workplace violence against homecare workers and its relationship with workers health outcomes: a cross-sectional study. BMC Public Health. 2015; 15(1): 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKowalenko T, Walters BL, Khare RK, et al.: Workplace violence: a survey of emergency physicians in the state of Michigan. Ann Emerg Med. 2005; 46(2): 142–7. PubMed Abstract | Publisher Full Text\n\nJoshua J: Gramling. Protecting the Protectors: Violence-Related Injuries to Hospital Security Personnel and the Use of Conducted Electrical Weapons. In partial fulfillment of the requirements; for the degree of doctor of philosophy. University of minnesota. 2017. Reference Source\n\nFindorff MJ, McGovern PM, Wall MM, et al.: Reporting violence to a health care employer: a cross-sectional study. AAOHN J. 2005; 53(9): 399–406. PubMed Abstract\n\nArnetz JE, Aranyos D, Ager J, et al.: Development and application of a population-based system for workplace violence surveillance in hospitals. Am J Ind Med. 2011; 54(12): 925–34. PubMed Abstract | Publisher Full Text\n\nPompeii L, Dement J, Schoenfisch A, et al.: Perpetrator, worker and workplace characteristics associated with patient and visitor perpetrated violence (Type II) on hospital workers: a review of the literature and existing occupational injury data. J Safety Res. 2013; 44: 57–64. PubMed Abstract | Publisher Full Text\n\nGreene J: Patient safety versus workplace safety: Stun gun debate illustrates dueling federal mandates. Ann Emerg Med. 2011; 57(4): A20–3. Publisher Full Text\n\nBastianelli BT: TASERS in healthcare: myths and merits. J Healthc Prot Manage. 2014; 30(1): 30–4. PubMed Abstract\n\nEl-Gilany AH, El-Wehady A, Amr M: Violence against primary health care workers in Al-Hassa, Saudi Arabia. J Interpers Violence. 2010; 25(4): 716–34. PubMed Abstract | Publisher Full Text\n\nPompeii LA, Schoenfisch AL, Lipscomb HJ, et al.: Physical assault, physical threat, and verbal abuse perpetrated against hospital workers by patients or visitors in six U.S. hospitals. Am J Ind Med. 2015; 58(11): 1194–204. PubMed Abstract | Publisher Full Text\n\nSchoenfisch AL, Pompeii LA: Security Personnel Practices and Policies in U.S. Hospitals: Findings From a National Survey. Workplace Health Saf. 2016; 64(11): 531–542. PubMed Abstract | Publisher Full Text\n\nArnetz JE, Hamblin L, Ager J, et al.: Application and implementation of the hazard risk matrix to identify hospital workplaces at risk for violence. Am J Ind Med. 2014; 57(11): 1276–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSullivan C, Yuan C: Workplace assaults on minority health and mental health care workers in Los Angeles. Am J Public Health. 1995; 85(7): 1011–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFernandes CM, Bouthillette F, Raboud JM, et al.: Violence in the emergency department: a survey of health care workers. CMAJ. 1999; 161(10): 1245–8. PubMed Abstract | Free Full Text\n\nLehmann LS, McCormick RA, Kizer KW: A survey of assaultive behavior in Veterans Health Administration facilities. Psychiatr Serv. 1999; 50(3): 384–9. PubMed Abstract | Publisher Full Text\n\nBensley L, Nelson N, Kaufman J, et al.: Injuries due to assaults on psychiatric hospital employees in Washington State. Am J Ind Med. 1997; 31(1): 92–9. PubMed Abstract | Publisher Full Text\n\nGerberich SG, Church TR, McGovern PM, et al.: An epidemiological study of the magnitude and consequences of work related violence: the Minnesota Nurses' Study. Occup Environ Med. 2004; 61(6): 495–503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrivitera M, Weisman R, Cerulli C, et al.: Violence toward mental health staff and safety in the work environment. Occup Med (Lond). 2005; 55(6): 480–6. PubMed Abstract | Publisher Full Text\n\nBehnam M, Tillotson RD, Davis SM, et al.: Violence in the emergency department: a national survey of emergency medicine residents and attending physicians. J Emerg Med. 2011; 40(5): 565–79. PubMed Abstract | Publisher Full Text\n\nArnetz J, Hamblin LE, Sudan S, et al.: Organizational Determinants of Workplace Violence Against Hospital Workers. J Occup Environ Med. 2018; 60(8): 693–699. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhittington R, Shuttleworth S, Hill L: Violence to staff in a general hospital setting. J Adv Nurs. 1996; 24(2): 326–33. PubMed Abstract | Publisher Full Text\n\nCamerino D, Estryn-Behar M, Conway PM, et al.: Work-related factors and violence among nursing staff in the European NEXT study: a longitudinal cohort study. Int J Nurs Stud. 2008; 45(1): 35–50. PubMed Abstract | Publisher Full Text\n\nShields M, Wilkins K: Factors related to on-the-job abuse of nurses by patients. Health Rep. 2009; 20(2): 7–19. PubMed Abstract\n\nGacki-Smith J, Juarez AM, Boyett L, et al.: Violence against nurses working in US emergency departments. J Nurs Adm. 2009; 39(7–8): 340–9. PubMed Abstract | Publisher Full Text\n\nFlannery RB Jr, Marks L, Laudani L, et al.: Psychiatric patient assault and staff victim gender: fifteen-year analysis of the Assaulted Staff Action Program (ASAP). Psychiatr Q. 2007; 78(2): 83–90. PubMed Abstract | Publisher Full Text\n\nKowalenko T, Gates D, Gillespie GL, et al.: Prospective study of violence against ED workers. Am J Emerg Med. 2013; 31(1): 197–205. PubMed Abstract | Publisher Full Text\n\nZhao S, Liu H, Ma H, et al.: Coping with Workplace Violence in Healthcare Settings: Social Support and Strategies. Int J Environ Res Public Health. 2015; 12(11): 14429–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalas MS, Kocaöz S, Akgüç S: A survey of violence against staff working in the emergency department in ankara, Turkey. Asian Nurs Res (Korean Soc Nurs Sci). 2011; 5(4): 197–203. PubMed Abstract | Publisher Full Text\n\nAhmed AA: Workplace violence against security personnel at a university hospital in Egypt. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/FRVSR2"
}
|
[
{
"id": "63327",
"date": "29 May 2020",
"name": "Ibrahim Kabbash",
"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\nThere is no need to mention the type of the study in the title.\n\nThe second paragraph in the introduction focused on health care violence. Security personnel can’t be considered as health care workers. The topic is violence in a health care setting rather than violence among health care workers. This issue should be considered in the whole part of introduction.\n\nThe authors did no mention the response rate among participants and whether there was any refusal to participate.\n\nWhat is the justification of categorization of participant by age at 35 years?\n\nIn table 4, event reporting to nursing syndicate is not logic and strange. It is logic to be reported to authority that can deal with the accident. What is the authority of nursing syndicates to deal with accidents occurring at the entrance of the hospital or the wards?\n\nIn table 5 how the exposed worker to violence feel guilty? I can understand he/she may feel ashamed but being guilty is not clear to me.\n\nIn discussion the authors repeated again most of figures mentioned in the results section. They should discuss information and not repeated results in discussion\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "65547",
"date": "14 Jul 2020",
"name": "Amal A. Hegazy",
"expertise": [
"Reviewer Expertise Occupational and environmental health topics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSecurity personnel are not considered one of the health care workers. The authors mentioned health care violence in the introduction, So, there is need to change.\n\nThe authors mentioned the response rate among the study group in the result section, it is better to transfer it to method section.\n\nIn logistic regression model, Kindly could the authors answer this question, what are the variables that entered in the model? In other words, did the authors make an association between the exposure and the outcome first , then he takes the significant one in the model or what did they do?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-347
|
https://f1000research.com/articles/9-345/v1
|
11 May 20
|
{
"type": "Opinion Article",
"title": "Recruiting the innate immune system with GM-CSF to fight viral diseases, including West Nile Virus encephalitis and COVID-19",
"authors": [
"Huntington Potter",
"Timothy D. Boyd",
"Penny Clarke",
"Victoria S. Pelak",
"Kenneth L. Tyler",
"Timothy D. Boyd",
"Penny Clarke",
"Victoria S. Pelak",
"Kenneth L. Tyler"
],
"abstract": "As the coronavirus disease 2019 (COVID-19) pandemic grows throughout the world, it is imperative that all approaches to ameliorating its effects be investigated, including repurposing drugs that show promise in other diseases. We have been investigating an approach to multiple disorders that involves recruiting the innate immune system to aid the body’s healing and regenerative mechanism(s). In the case of West Nile Virus encephalitis and potentially COVID-19, the proposed intervention to stimulate the innate immune system may give the adaptive immune response the necessary time to develop, finish clearing the virus, and provide future immunity. Furthermore, we have found that GM-CSF-induced recruitment of the innate immune system is also able to reverse brain pathology, neuroinflammation and cognitive deficits in mouse models of Alzheimer’s disease and Down syndrome, as well as improving cognition in normal aging and in human patients with cognitive deficits due to chemotherapy, both of which exhibit neuroinflammation. Others have shown that GM-CSF is an effective treatment for both bacterial and viral pneumonias, and their associated inflammation, in animals and that it has successfully treated pneumonia-associated Acute Respiratory Distress Syndrome in humans. These and other data strongly suggest that GM-CSF may be an effective treatment for many viral infections, including COVID-19.",
"keywords": [
"COVID-19",
"SARS-CoV-2",
"West Nile Virus",
"granulocyte-macrophage colony-stimulating factor",
"Alzheimer’s Disease",
"inflammation",
"acute respiratory distress syndrome",
"neuroinflammation"
],
"content": "\n\nThere is much debate about how to best treat disorders that include an inflammatory component. For example, in Alzheimer’s disease (AD), inflammation is evidenced by the activation of microglia and astrocytes, the consequent over-expression of inflammatory cytokines and acute phase proteins in the brain, and by the presence of inflammatory markers in the blood1,2. Furthermore, in AD, some inflammatory proteins expressed in the brain, specifically α1-antichymotrypsin and apolipoprotein E (ApoE), especially ApoE4, clearly promote and, indeed, are necessary for amyloidogenesis that leads to cognitive decline in humans and/or animal models3,4. The finding that the introduction of pro-inflammatory molecules, such as lipopolysaccharide, into mouse models of AD exacerbated amyloid pathology and cognitive deficits also suggested that inflammation contributed essentially to the disease, possibly by a self-perpetuating feed-forward mechanism5. This idea was reinforced by the finding that people with rheumatoid arthiritis, who routinely take non-steroidal anti-inflammatory drugs (NSAIDS) to control the swelling and inflammation in their joints, have a much lower risk of developing AD, which suggested that their NSAID use was protective against, and might be used to treat, AD6,7.\n\nIn the case of viral diseases, including West Nile Virus (WNV) encephalitis, bacterial and viral pneumonia, Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV-1), Middle East Respiratory Syndrome-coronavirus (MERS-CoV), the development of inflammation has been cited as evidence that an effective treatment should include anti-inflammatory drugs, including steroids8,9. Indeed, steroids have been used in some human patients with WNV infection10–13. In respiratory diseases, an overactive innate immune reaction, termed a ‘cytokine storm’ may arise and has been treated with high dose steroids. Furthermore, use of minocycline to suppress microglial activation and promote expression of anti-inflammatory cytokines reduces neuronal cytotoxicity and mortality in WNV-infected mice14,15. Minocycline, has also been suggested as a treatment for AD based on experiments in cell culture and animal models, but its effects were complex and resulting changes to the peripheral and CNS immune system remain unclear16,17.\n\nDespite the logical and preclinical data-based arguments in favor of using anti-inflammatory drugs for treatment of inflammation-associated disorders, clinical trials showed that NSAIDs were unsuccessful and potentially detrimental in AD and mild cognitive impairment participants7,18–20, and minocycline also failed in a recently-completed trial in participants with early AD21. In WNV and Japanese Encephalitis Virus infected mice, reducing microglia in the brain by blocking the receptor of Macrophage Colony Stimulating Factor (MCSF) with PLX56229 resulted in increased viral load and mortality, suggesting that microglia, traditionally considered to be an indication of a dangerous pro-inflammatory milieu may, in fact be beneficial in fighting the virus infection.\n\nSuch alternative, even opposing, views of the role of inflammation is particularly striking in recent discussions about treating COVID-19. For example, it is proposed that inflammation in COVID-19-associated SARS should be suppressed because the cytokine storm is thought to play a major role in the pulmonary damage that so greatly increases morbidity and mortality in SARS (also termed acute respiratory distress syndrome, ARDS), a feature of infection by both SARS-CoV-1 and SARS-CoV-222. Recent studies report increased plasma levels of chemokines and cytokines, including CCL-2, CCL-3, RANTES, INFγ, IL-1β, IL-2, IL-6, IL-8, IL-10, G-CSF, IP10, TNFα, and others, in severe cases of COVID-19 as compared to mild cases23,24, while decreased levels of lymphocytes are also commonly found in severe cases of SARS and COVID-19 patients25–30. Thus, it is unclear whether treatment with broad-acting immunosuppressant drugs to combat the increased cytokine and chemokine signaling would also exacerbate leukopenia, resulting in inhibited clearance of the virus and repair of tissue damage. Because no controlled trials using NSAIDs or steroids in COVID-19 patients have yet been reported, the CDC has taken a cautious stand, stating that “Currently, there are no data suggesting an association between COVID-19 clinical outcomes and NSAID use” and that “Corticosteroids have been widely used in hospitalized patients with severe illness in China31–34; however, the benefit of corticosteroid use cannot be determined based upon uncontrolled observational data. By contrast, patients with MERS-CoV or influenza who were given corticosteroids were more likely to have prolonged viral replication, receive mechanical ventilation, and have higher mortality35–39. Therefore, corticosteroids should be avoided unless indicated for other reasons, such as management of chronic obstructive pulmonary disease exacerbation or septic shock.”40\n\nAs another, more targeted approach, it is suggested that inflammation may be best prevented or reduced by using specific monoclonal antibodies to block either the IL-6 receptor41–43 or the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF)41,44. The hypothesis that blocking IL-6 or GM-CSF will be beneficial is based in part on the finding that IL-6 and GM-CSF and/or the cells that express them are increased in the blood of COVID-19 patients22,41 and on the beneficial results froma preliminary single-arm trial of an IL-6 receptor blocker, which reportedly lowered fever and improved oxygenation in COVID-19 patients43. Lowering IL-6 or other inflammatory cytokines in COVID-19 patients is reasonable, but the timing may be critical as early treatment may be detrimental. A multi-arm study including both early and later stage patients is essential to determine the clinical relevance of such intervention30,42, especially for anti-IL-6 therapies, such as tocilizumab and sarilumab, as IL-6-mediated inflammation has been shown to play a critical role in effective cellular and humoral immunity against viral infections45–48. Thus, if anti-IL-6 therapies are used too early to treat patients with COVID-19, this strategy could potentially inhibit viral clearance and/or leave the patients susceptible to recurrent infection with SARS-CoV-2.\n\nAs an alternative to treatment approaches designed to block pro-inflammatory cytokine responses, we suggest that COVID-19-associated SARS may be better prevented or treated by instead increasing the activation and numbers of macrophages and microglia, in particular by increasing the levels of GM-CSF. This innate immune system stimulant has been approved by the FDA as recombinant human GM-CSF (rHuGM-CSF/sargramostim/Leukine®) for almost 30 years as a therapy to increase white blood cell numbers by stimulating the division and differentiation of hematopoietic stem cells49. Approved indications for such treatment include reducing the incidence of severe and life-threatening infections after chemotherapy, accelerating myeloid reconstitution following hematopoietic cell transplantation (HCT), and treating cases of acute radiation syndrome. The finding of low white blood cell counts in patients with COVID-1923 suggests that the ability of GM-CSF to increase select leukocyte populations, particularly phagocytes, may be of therapeutic value in COVID-19, especially given GM-CSF’s ability to treat life-threatening infections, and as data from Wuhan, China have shown that half of all COVID-19 patients who died had evidence of secondary infections50. This concept is further supported by a previous study that found that the endogenous levels of GM-CSF were significantly higher in bronchoalveolar lavage fluid from patients with ARDS due to trauma or sepsis who survived compared to those who died51, in contrast to the potential deleterious effects of GM-CSF that were inferred from the study cited above41.\n\nThese results suggest to us that GM-CSF may be beneficial in the treatment of WNV infection and the inflammation that results, and by implication, other viral infections, potentially including COVID-19. In preliminary experiments, we have found that GM-CSF treatment improves survival in mice infected by footpad injection with the TX02 strain of WNV, from approximately 35% in untreated animals to 80% survival in mice treated with GM-CSF starting the day after viral inoculation (p=0.0034) (Clarke, Potter, Boyd, Tyler, manuscript in preparation). These findings are particularly important, as there are currently no effective treatments for WNV encephalitis, and the long safety history of rHuGM CSF/sargramostim in treating leukopenia and preventing life-threatening infections suggests that these preliminary findings, if confirmed, could quickly be translated into human trials.\n\nFurthermore, the potential benefit of GM-CSF as a pulmonary therapy has been shown directly in several other animal models in which GM-CSF treatment was able to both protect against and treat bacterial and viral pneumonias52–61, which, like COVID-19, induce a cytokine storm that can lead to respiratory distress and multi-organ failure. Notably, some of these studies showed that inhaled GM-CSF was particularly efficacious57,58. Inhaled GM-CSF has also been used successfully in humans off-label as a treatment for pulmonary alveolar proteinosis62–65, and successfully as compassionate treatment for pneumonia-associated ARDS66.\n\nThe recent report that injection of mesenchymal stem cells (MSCs) reverses clinical and peripheral manifestations of COVID-1967 and the fact that GM-CSF is well known to stimulate the mobilization of MSCs from the bone marrow68–70 provide further support for the argument that GM-CSF administration may effectively treat COVID-19.\n\nIt is also relevant that the unusually rapid onset of COVID-19-associated SARS after infection with the SARS-CoV-2 virus may be due to infection and inflammation in the central nervous system (CNS), including in the brain stem, which could contribute to respiratory failure, and neurological symptoms have been reported in COVID-19 patients, such as hypogeusia, hyposmia, neuralgia, dizziness, headache, ataxia, epilepsy, and others71–77. Additionally, in studies of SARS-CoV-1 and MERS-CoV, the presence of viral neuroinvasion has been identified in both animal studies78,79 and in post-mortem human brain samples80,81. Significantly, GM-CSF can easily cross the blood-brain barrier82, and our work with GM-CSF in both animals and humans indicates that it can ameliorate CNS disorders of various kinds that include an inflammatory component. For example, GM-CSF/sargramostim treatment is associated with improved cognition in:1) mouse models of Alzheimer’s disease83, which has since been replicated by others84, 2) a mouse model of Down syndrome (Ahmed, Boyd, Potter et al., manuscript in preparation), 3) aged wild-type control mice83 and (Ahmed, Boyd, Potter et al., manuscript in preparation), which has also been replicated by others85, 4) a retrospective study of leukemia patients after immune system chemoablation and HCT, who acquire chemotherapy-associated cognitive impairment86, and 5) mild-to-moderate Alzheimer’s disease participants, based on one measure, the Mini-Mental State Exam (recombinant human GM-CSF/sargramostim, five days/week for three weeks (Potter, Woodcock, Boyd et al., NCT01409915; manuscript submitted), all of which are CNS disorders. These findings are relevant to COVID-19 because all of these disorders induce inflammation1,8,9 and yet still benefit from treatment with GM-CSF, which increases, rather than decreases, the number and activation of microglia, the resident phagocytes of the CNS that are often cited as indicators of a dangerous pro-inflammatory milieu that must be suppressed to achieve successful treatment. Additionally, we have found that GM-CSF treatment reverses astrogliosis, which is also associated with CNS disorders and has been reported to be reversed by GM-CSF treatment that ameliorates spinal cord injury and inhibits glial scar formation87,88. Furthermore, multiple studies have shown that there is a very high prevalence of cognitive impairment in ARDS survivors, from between 70–100% at hospital discharge, to 46–80% at one year, and persisting in 20% at five years89–91, which strongly argues for extended treatment with GM-CSF/sargramostim, given its ability to improve cognition in several animal models and reverse cognitive impairment associated with chemotherapy and neurodegeneration.\n\nBecause GM-CSF/Sargramostim has been safely and routinely used as a subcutaneous injection treatment and, off-label, as an inhalation treatment, we suggest that this long-FDA-approved drug should be tested for its ability to improve recovery in COVID-19 patients and/or after resolution of infection in severe ARDS survivors to ameliorate potential cognitive deficits. Although compassionate use is possible, it is more important that, either, or a combination, of these two routes of administration in a randomized, pilot trial be used to properly assess the possibility that the innate immune system stimulant, GM-CSF, may reduce morbidity and mortality from COVID-19, as the studies discussed above suggest. Indeed, while an earlier version of this manuscript was being reviewed, a trial of sargramostim in COVID-19 patients with ARDS was announced in Europe using both inhaled/nebulized and/or injected administrations92. We hope that sites in the U.K., the U.S., and elsewhere will be invited to join that study, and welcome comments and data at: https://medschool.cuanschutz.edu/GMCSF-COVID\n\nIf GM-CSF/sargramostim proves to be beneficial as a treatment for COVID 19, the fact that the U.S. Department of Health and Human Services maintains a stock of rHuGM-CSF/sargramostim to be used in the event of a nuclear accident may provide short-term resources93.\n\n\nData availability\n\nNo data is associated with this article.",
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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPartner Therapeutics Announces Initiation of Clinical Trial to Evaluate Leukine® in Patients with COVID-19 Associated Respiratory Illness. 2020. Reference Source\n\nASPR: BARDA FDA 50 Product Approvals - Alternative Text Description. 2019. Reference Source"
}
|
[
{
"id": "63768",
"date": "23 Jun 2020",
"name": "Sharon Celeste Morley",
"expertise": [
"Reviewer Expertise Pulmonary innate immunity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion article by Potter et al. presents the provocative hypothesis that targeted stimulation of certain immune populations by the administration of GM-CSF could ameliorate certain neuroinflammatory conditions, such as Alzheimer's or West Nile Virus infection, or pulmonary conditions such as COVID.\nThere are many references drawn together to support this suggestion. However, there is insufficient clarity in the arguments to introduce the subject to readers unfamiliar with the literature.\nOf largest concern is the casual mixing of very different immunomodulators, without discussion of the various mechanisms of action. The authors discuss steroids, NSAIDS, minocycline, monoclonal antibody therapies, and an experimental therapy called PLX5622 without an adequate introduction to the very different pathways inhibited by these modulators.\nAdditionally, the authors do not always distinguish between the different cell types affected. For instance, they comment that GM-CSF might help COVID by improving the \"leukopenia\" noted in severe cases. However, it is LYMPHOPENIA that marks the most severe cases of COVID, and systemic GM-CSF administration will not help that at all.\nIn terms of GM-CSF, the authors do not clearly distinguish between the likely different mechanisms of action when GM-CSF is administered systemically vs via inhalation. It is unlikely, for instance, that inhaled GM-CSF stimulates demargination of PMNs to the same extent that subQ does.\nFinally, the combined discussions of auto-neuro-inflammation (e.g. Alzheimer's), WNV neuroinflammation, and pulmonary inflammation becomes confusing.\nIn short, this article would be vastly improved by separating the topic into multiple subheadings, and addressing one sub-topic (e.g. \"Immunomodulators in Alzheimer's,\" \"Immunomodulators in WNV,\") at a time.\nThe information is good. The references are solid. The proposal of using GM-CSF therapeutically to modify inflammation is exciting. But the disorganization of this article currently obscures its strengths, and it should be rewritten for clarity.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "63287",
"date": "10 Jul 2020",
"name": "W.Sue T Griffin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn an interesting perspective on immune system-based strategies to combat COVID-19 and its accompanying cytokine storm, Potter et al. discuss the relationship of this disease—and particularly, the hyperinflammatory syndrome manifest in the most severe cases—to other disorders. While it may not be surprising to SARS-CoV-2 infections placed vis-à-vis with other viral infections such as West Nile Virus (WNV), some readers may be shocked to see the comparison extended to Alzheimer’s disease (AD). But this is not an unreasonable stretch. The inflammatory hypothesis of AD has been around since the late 1980s, when we and others began to report the elevation of immune-system markers such as interleukin-1 and complement proteins1'2'3'4. We were reluctant to refer to these changes as “inflammation” at the time, as the brain’s partial privilege from immune sequelae5 puts a damper on the calor, dolor, and rubor classically associated with peripheral inflammation. Nevertheless, the cytokines most closely associated with inflammation now appear to drive every element of AD pathology, as we proposed in the “cytokine cycle”6 (which a more poetic turn of phrase might have rendered “cytokine cyclone” and thus provided the first such “storm”).\n\nThe elaboration of cytokines during neurodegeneration must be presumed to arise from some program that would be otherwise appropriate and adaptive, and several findings indicate that it is modulation rather than wholesale suppression of immune mediators that will be beneficial in AD. Potter et al. touch on this in reference to the effects of lipopolysaccharide in mouse models of AD, though they could have helped their case by including the intriguing finding that such stimuli can attenuate amyloid pathology7. Many current therapeutic attempts are aimed at manipulating inflammatory pathways in AD, including the application of GM-CSF (or a clinical preparation known as “sargramostim”) by Potter’s group. The strange career of COVID-19 and the varied effects it has on multiple organ systems suggests a similar need for targeted manipulation of immune responses.\n\nEvidence clearly indicates that SARS-CoV-2 promotes inflammation by augmenting actions of the innate immune system. In addition to the cytokine storm, the life-threatening form COVID-19 sometimes takes is associated with other inflammatory markers such as C-reactive protein, ferritin, and D-dimers, as well as elevation of the neutrophil:lymphocyte ratio8. It is possible that this reflects attempts by the innate immune system to pick up the slack left by virus’s targeted suppression of acquired immunity via an exquisite ability to suppress interferon-dependent responses9. This provides one more similarity to AD via the changes in immune systems brought about by advanced age, which invariably results in lymphocyte decline and the elevation of inflammatory cytokines as a component of the senescence-associated secretory phenotype.\n\nPotter et al. acknowledge this evidence of an overactive innate response in their criticism of treatments that would suppress the entire immune system, such as steroids and NSAIDS. This latter is also cited in relation to the ADAPT trial of naproxen or celecoxib in AD, halted due to untoward side effects of celecoxcib. However, it is interesting to note, as the authors do, that nondemented subjects who took naproxen for two or three years of the trial had reduced rates of developing AD. Such findings fit well with a VA database retrospective study of ibuprofen and naproxen use among almost 50,000 clinically and neuropathologically confirmed Alzheimer patients and 200,000 non-AD patients. The odds of being in the AD group was reduced from 1.03 to 0.56—i.e., more than 40 percent—among those who took ibuprofen for 5 years or longer10.\n\nLess compelling are the prospects for effectively guiding the immune system through a SARS-CoV-2 attack with GM-CSF. Potter et al. suggest that the success of sargramostim to treating some forms of leukopenia means it will boost lymphocyte actions leukopenia. However, the difference in CD4+ lymphocytes produced by sargramostim treatment was just 22 percent, and this was only after 6 months of treatment11. The vast majority of leukocytes that accumulate after GM-CSF/sargramostim administration are neutrophils, with a considerable number of eosinophils, monocytes, and macrophages; this would seem to be precisely contraindicated in COVID-19. Indeed, Zhou et al. (2020), cited by Potter et al., suggest suppression of GM-CSF among other strategies to restrain the innate immune system.\nThus, the logical argument for GM-CSF in SARS or COVID-19 is one that should raise red flags; there is a chance that it could worsen outcomes. Improved outcomes with GM-CSF in the WNV model is encouraging. However, it should be noted that WNV is in an entirely different phylum from coronaviruses. Thus, the WNV data may serve best as justification for conducting preclinical studies with a relevant coronavirus model. In the end, it is imperative that no stone is left unturned in the quest for effective treatments for COVID-19 while we suffer without a vaccine. For regardless of how different that acute disease might be from AD and related disorders, there is every reason to believe that the inflammatory ravages triggered by SARS-CoV-2 will set in motion cyclones and cycles that will increase the rates of chronic neurodegeneration.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "65612",
"date": "13 Jul 2020",
"name": "Zissis Chroneos",
"expertise": [
"Reviewer Expertise Mechanisms of infectious and noninfectious inflammatory syndromes of the lung",
"surfactant protein immune functions",
"GM-CSF in pulmonary homeostasis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe commentary of Potter et al. draws parallels from their finding that GM-CSF ameliorates intrinsic and viral neuro-inflammation induced in the course of Alzheimer's disease and West Nile Virus infection, respectively. The authors propose that the application of GM-CSF may have utility for the treatment of COVID-19. Neurotropism for SARS-CoV-2 and other respiratory coronaviruses has been documented, although this is not discussed in the present commentary as a relevant source of targetable neuroinflammation by GM-CSF administration.\nSupporting a beneficial role of GM-CSF, there is an abundance of studies that supplementation of GM-CSF in the airway enhances resistance to viral and bacterial pneumonia and, furthermore, that therapeutic GM-CSF inhalation can stabilize critical illness by macrophages increasing the odds for survival. These beneficial effects are observed locally at a high GM-CSF dosage. It is well-established that the effects of GM-CSF on myeloid cell behavior, activation, and interactions interfacing innate and adaptive immunity are concentration- and source-dependent. Homeostatic functions of GM-CSF at local sites, such as surfactant metabolism by alveolar macrophages may contribute to anti-inflammatory scavenging and repair of tissue damage at a time of need. On the other hand, activated lymphocytes unloading their GM-CSF payload in inflammatory immune synapses during immune remediation against an infectious agent could deliver substantial collateral damage and tissue destruction. To this end, there is an abundance of studies that GM-CSF contributes to adverse inflammation in multiple sterile and non-sterile inflammatory models, autoimmune disease models, and in chronic inflammatory disease caused by chronic environmental exposures such as smoking.\nButtressing a detrimental contribution of GM-CSF in the development of COVID-19, there are a plethora of clinical trials underway to neutralize GM-CSF signaling. This, however, is evolving against a backdrop of disappointing data that IL-6 blockade in the Sanofi and Regeneron trials were not effective for the treatment of COVID-19. The article could be updated with this latest information, although the effects of GM-CSF in such trials remains to be seen.\nThe challenge in designing immuno-modulatory therapies for COVID-19 is the systemic nature of the syndrome. Timing, disease stage, manifestation, and severity, dosage, and route of administration are critical parameters. Significant research effort is, therefore, required to inform the decision making process for GM-CSF supplementation or blockade given the diverse functions of resident myeloid cell populations in different tissues. A more discerning argument on the compartmental utility of GM-CSF in preventing cognitive sequelae of COVID-19 is thus warranted.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-345
|
https://f1000research.com/articles/9-343/v1
|
11 May 20
|
{
"type": "Case Report",
"title": "Case Report: Metastatic breast cancer to the gallbladder",
"authors": [
"Giulia Missori",
"Francesco Serra",
"Giorgia Prestigiacomo",
"Andrea Aurelio Ricciardolo",
"Lucio Brugioni",
"Roberta Gelmini",
"Giulia Missori",
"Francesco Serra",
"Giorgia Prestigiacomo",
"Andrea Aurelio Ricciardolo",
"Lucio Brugioni"
],
"abstract": "Cholecystitis is one of the leading causes of emergency surgical interventions; the occurrence of metastases to the gallbladder is rare and has only been reported in the literature exceptionally. Metastatic breast cancer to the gallbladder is even less frequent; in fact, breast cancer usually metastasizes to bone, lung, lymph nodes, liver and brain. We report the case of an 83-year-old female patient with a previous history of breast surgery with axillary dissection in 1997, followed by adjuvant chemotherapy due to invasive ductal carcinoma of the left breast. The patient was admitted at the emergency department for sepsis and an episode of acute kidney failure, anuria and fever. Right-upper quadrant abdominal pain triggered by food intake and abdominal tenderness was also present, placing the diagnostic suspicion of biliary sepsis due to acute cholecystitis. The histological examination of the surgical specimen highlighted the presence of metastasis from an infiltrating ductal breast carcinoma with positive hormone receptors. We also report here the results of a review of the literature looking at articles describing cases of gallbladder metastasis from breast cancer.",
"keywords": [
"Emergency surgery",
"Breast cancer",
"Cholecystitis"
],
"content": "Introduction\n\nCholecystitis is one of the leading causes of emergency surgical interventions. The diagnosis of acute cholecystitis is usually based on physical examination, laboratory tests and abdominal ultrasound. The surgical options for cholecystitis are either open and laparoscopic cholecystectomy; the latter is nowadays considered the gold standard of treatment. Surgical specimens must be sent for histopathological examination to rule out cancer1.\n\nThe occurrence of metastases to the gallbladder is rare and has only been reported in the literature exceptionally2. Primary tumors can metastasize to the gallbladder either by proximity, such as hepatocellular carcinoma and pancreatic carcinoma, or by blood diffusion3.\n\nChan reported, in a series of 7910 cholecystectomy specimens, that 36 cases of metastatic carcinoma were found, more often secondary to the stomach, lower gastrointestinal tract, liver, kidney or skin (malignant melanoma) cancer4. Another more recent study shows that metastasis to the gallbladder accounted for 7/225 (3.1%) of the incidental gallbladder malignancies5. Metastasis from breast cancer to the gallbladder is even less common; in fact, breast cancer usually metastasizes to bone, lung, lymph nodes, liver and brain.\n\nWe describe here the case of a patient who underwent cholecystectomy for acute cholecystitis with gallbladder metastasis from breast cancer. Subsequently, we present the results of a literature search concerning this disease.\n\n\nCase report\n\nWe report the case of an 83-year-old female patient with a previous history of breast surgery with axillary dissection in 1997, followed by adjuvant chemotherapy due to invasive ductal carcinoma of the left breast. The family history was negative for neoplastic diseases, both mammary and belonging to the gastrointestinal tract. Oncological follow-up was negative, and the patient considered disease-free for almost 15 years. During 2012, an X-ray of the spine, performed for the appearance of lumbar pain, revealed the presence of vertebral metastases. The patient was treated with radiotherapy and spinal stabilization. In addition to this, a deep venous thrombosis episode was reported in 2017, and treated with anticoagulant therapy. In the same year, multiple myeloma associated with mild chronic kidney disease was diagnosed. Neither myeloma nor kidney disease had requested specific treatments.\n\nIn July 2018, the patient was admitted to the emergency department for sepsis and an episode of acute kidney failure, anuria and fever. Right-upper quadrant abdominal pain triggered by food intake and abdominal tenderness was also present, placing the diagnostic suspicion of biliary sepsis due to acute cholecystitis.\n\nThis condition was conservatively treated with intravenous antibiotic therapy with renal adjusted dose of piperacillin-tazobactam and hemodialysis for two weeks. Subsequently, kidney function improved, diuresis had an increasing glomerular filtration rate and sepsis was cured. Abdominal CT-scan performed during this hospitalization had shown a diffuse thickening of the gallbladder’s wall associated with stones as well as pericholecystic fluid (Figure 1). The CT-scan didn’t highlight pathological findings on the liver, such as enlarged regional nodes. A dilated common bile duct with the presence, in its proximal portion, of tenuously hyperdense material was described.\n\nEndoscopic ultrasound was performed, and it confirmed the presence of both gallbladder and common duct stones, the largest was 7 millimetres, and biliary sludge with lack of dilatation of the intrahepatic biliary tract. Several stones were removed via endoscopic retrograde cholangiopancreatography, and a nasobiliary tube was left behind. Subsequent cholangiography demonstrated the regular calibre and morphology of the cystic duct, the principal biliary tract, and the intrahepatic biliary tree. However, the gallbladder appeared distended with several little stones inside.\n\nThe patient, after 6 days from the admission, finally underwent laparoscopic cholecystectomy. Intraoperative findings showed the gallbladder with thickened walls and densely fused with the liver but without other pathological findings. No intraoperative complications occurred. Histological examination of the surgical specimen highlighted the presence of metastasis from an infiltrating ductal breast carcinoma with positive hormone receptors: Estrogen Receptors (MoAb SP1) 98%, Progesterone Receptors (MoAb 1E2) 95%, Cytoprolferative Activity (MoAb MIB-1) 10%, c-erbB2 (MoAb 4B5) score: 0. The cystic lymph node showed no evidence of metastasis. The postoperative course was regular, and the patient was transferred to a rehabilitation ward five days after surgery.\n\nAfter completion of the rehabilitation program, the patient was discharged, and hormone therapy (letrozole 2.5 mg once a day) was started. The patient died 15 months later due to peritoneal and bone progression of the disease.\n\n\nReview of the literature\n\nWe conducted a systematic review in which all articles describing cases of gallbladder metastasis from breast cancer were considered eligible for inclusion. Abstracts, conference papers and studies concerning animals were excluded. No restrictions were applied to publication date or languages, if there was an English version of the article available.\n\nA systematic search for articles published up to February 2020 using PubMed, Scopus, Google Scholar and Web of Science databases was performed, and references of articles that were retrieved in the full text were also searched. The search strategy utilized in all databases included the combination of the keywords: “gallbladder metastasis”, “breast cancer”, “acute cholecystitis”, “biliary colic”, “cholelithiasis”. A minimum number of two search keywords were utilized, one of which was always “breast cancer”.\n\nA total of 848 potentially relevant articles were retrieved in Google Scholar, 427 in Scopus, 182 in Web Of Science and 123 in PubMed. Among these 22 studies were identified to be strictly matched with our research (Figure 2). Our case was also included in the review.\n\n\nDiscussion\n\nIn consequence of advances in medical chemotherapy and endocrine therapy in the last years, the outcomes for breast cancer are improved. Disease recurrence is more common within five years of surgery while late recurrences after more than 10 years are very uncommon. The literature outlines risk factors for late recurrence as lymph node metastases, ER + status and HER-2 negative status6,7. Breast cancer metastases occur through contiguous, lymphatic and hematogenous spread. It usually metastasizes to bone, lung, lymph nodes, liver and brain. Less frequently invaded are the endocrine organs, pericardium, abdominal cavity and eyes. Metastasis in the extrahepatic digestive system are infrequent and characteristically appear after a long latent period, which takes from three to up to 20 years5.\n\nConcerning gallbladder metastases by breast cancer, autopsy findings have shown that secondary hematogenous metastases (also from other primary organs) to the gallbladder initially generate small flat nodules below the mucosal layer. They grow as a pedunculated tumor, rarely reaching higher than several millimetres in size. The growth pattern clarifies why gallbladder metastases rarely result in clinical symptoms and that they are not diagnosed during patients’ lives. Metastatic gallbladder tumors rarely show signs; acute cholecystitis is the most frequent clinical presentation8. Obstructive jaundice, haemobilia, even bile peritonitis due to perforation, are seldom described. When a gallbladder metastasis is identified after surgery, the primary tumor can be not easily defined. Distinguishing between primitive gallbladder carcinoma and metastases from breast cancer is crucial for proper post-surgery therapy; in this way, immunohistochemical evaluation is necessary. The most reliable markers are gross cystic disease fluid protein such as 15 (GCDEP -15), plus cytokeratin 7, cytokeratin 20, and estrogen and progesterone receptors. Usually, their positivity is present in metastatic breast cancer, but not in all cases9.\n\nAt microscopic pathological examination, metastases are often represented by small clusters and chains of neoplastic cells, commonly of the signet-ring histotype. Pathological diagnosis of metastases from lobular breast cancer can be difficult because signet-ring cells could be present in tumors originating from different organs, such as the stomach10.\n\nOur review of the literature conducted on secondary lesions of the gallbladder from breast cancer has confirmed the rarity of this disease (see Table 1 for a summary of the cases). Gallbladder metastasis is only described in 23 patients, including our case: 11 from infiltrating lobular, 7 ductal origins, 3 mixed ductal and lobular infiltration, and 3 not specified. This analysis reveals how, in most cases (12), the diagnosis of metastatic lesions was made after surgery was performed for acute cholecystitis. There was evidence of gallstones in 8 cases; 9 cases were patients who often suffer from abdominal pain and/or vomiting (symptoms of biliary colic), and so they underwent an elective cholecystectomy. Only in 2 cases, the main symptom was obstructive jaundice or bile peritonitis for necrotic gallbladder.\n\nInstrumental diagnostics are useless as they do not show significant data on gallbladder walls that are suspicious for malignancy; the identification of the neoplastic disease is possible only after surgery during histological examination of the specimen, as was shown in our case. From the analysis of the cases described in the literature, it follows that the most frequent tumor histology associated with gallbladder metastasis by breast cancer is infiltrating lobular carcinoma.\n\nThis review shows how the detection of gallbladder metastasis usually occurs any time after the surgery for the primary tumor. In essence, we would highlight that in 6 cases, it happened after more than 10 years from primary surgery, in 7 cases between 1 and 6 years, and 3 cases within the first year. Only in 6 cases was the detection of breast cancer and gallbladder metastasis synchronous.\n\n\nConclusions\n\nThis report emphasizes the importance of long-term follow up in patients with a history of breast cancer.\n\nOur experience and data from the literature suggest carefully evaluating every anomaly observed during routine staging examinations, even when apparently due to benign, mild disease. Metastatic disease always should be included in the differential diagnosis of a patient with a history of invasive breast cancer and new onset of abdominal pain. Conventional methods of documenting gallbladder disease are nonspecific concerning the malignant disease. This may pose a diagnostic challenge in patients with abdominal symptoms after resection of malignancies, also because they need to be aggressively treated as it can improve the poor prognosis of these cases. From our case and literature review, we recommend the following:\n\n1. Consider the oncological story of the patients in the emergency setting;\n\n2. Metastatic disease should be included in the differential diagnosis in patients with a history of breast cancer.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient on admission to hospital prior to the patient’s death.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "References\n\nYoon WJ, Yoon YB, Kim YJ, et al.: Metastasis to the gallbladder: a single-center experience of 20 cases in South Korea. World J Gastroenterol. 2009; 15(38): 4806–4809. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuszynska C, Lundgren L, Andersson R, et al.: Incidental metastases and lymphoma of the gallbladder - an analysis of ten rare cases identified from a large national database. Scand J Gastroenterol. 2019; 54(3): 350–358. PubMed Abstract | Publisher Full Text\n\nTerasaki S, Nakanuma Y, Terada D, et al.: Metastasis of hepatocellular carcinoma to the gallbladder presenting massive intraluminal growth: Report of an autopsy case. J Clin Gastroenterol. 1990; 12(6): 714–5. PubMed Abstract | Publisher Full Text\n\nChan KW: Review of 253 cases of significant pathology in 7,910 cholecystectomies in Hong Kong. Pathology. 1988; 20(1): 20–3. PubMed Abstract | Publisher Full Text\n\nMurguia E, Quiroga D, Canteros G, et al.: Gallbladder metastases from ductal papillary carcinoma of the breast. J Hepatobiliary Pancreat Surg. 2006; 13(6): 591–3. PubMed Abstract | Publisher Full Text\n\nNishimura R, Osako T, Nishiyama Y, et al.: Evaluation of factors related to late recurrence—later than 10 years after the initial treatment—in primary breast cancer. Oncology. 2013; 85(2): 100–10. PubMed Abstract | Publisher Full Text\n\nVen Ustaalioglu BB, Balvan O, Bilici A, et al.: The differences of clinicopathological factors for breast cancer in respect to time of recurrence and effect on recurrence-free survival. Clin Transl Oncol. 2015; 17(11): 895–902. PubMed Abstract | Publisher Full Text\n\nBush E, Geradts J, O'Connor T: An unusual cause of cholecystitis? Am J Clin Oncol. 2005; 28(5): 529–30. PubMed Abstract | Publisher Full Text\n\nManouras A, Lagoudianakis EE, Genetzakis M, et al.: Metastatic breast carcinoma initially presenting as acute cholecystitis: a case report and review of the literature. Eur J Gynaecol Oncol. 2008; 29(2): 179–81. PubMed Abstract\n\nMerino MJ, Livolsi VA: Signet ring carcinoma of the female breast: A clinicopathologic analysis of 24 cases. Cancer. 1981; 48(8): 1830–7. PubMed Abstract | Publisher Full Text\n\nDi Vita M, Zanghì A, Lanzafame S, et al.: Gallbladder metastases of breast cancer: from clinical-pathological patterns to diagnostic and therapeutic strategy. Clin Ter. 2011; 162(5): 451–6. PubMed Abstract\n\nBeaver BL, Denning DA, Minton JP: Metastatic breast carcinoma of the gallbladder. J Surg Oncol. 1986; 31(4): 240–2. PubMed Abstract | Publisher Full Text\n\nShah RJ, Koehler A, Long JD: Bile peritonitis secondary to breast cancer metastatic to the gallbladder. Am J Gastroenterol. 2000; 95(5): 1379–81. PubMed Abstract\n\nRubin A, Tate JJ: Breast carcinoma metastatic to the gallbladder. J Clin Pathol. 1989; 42(11): 1223–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHashimoto M, Koide K, Arita M, et al.: Acute acalculous cholecystitis due to breast cancer metastasis to the cystic duct. Surg Case Rep. 2016; 2(1): 111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nColetta M, Montalti R, Pistelli M, et al.: Metastatic breast cancer mimicking a hilar cholangiocarcinoma: case report and review of the literature. World J Surg Oncol. 2014; 12: 384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNair MS, Phillips BL, Jain G, et al.: Gall Bladder Metastasis from Breast Cancer Masquerading Symptomatic Cholelithiasis. J Gastrointest Cancer. 2012; 43 Suppl 1: S215–6. PubMed Abstract | Publisher Full Text\n\nAl-Rawi H, Al-Jafari M, Mathew H: Metastatic breast cancer mimicking cholecystitis. A rare clinical presentation. Saudi Med J. 2012; 33(10): 1128–30. PubMed Abstract\n\nEbrahim H, Graham D, Rice D, et al.: Inflammatory metastatic breast cancer with gallbladder metastasis: an incidental finding. J Community Support Oncol. 2015; 13(7): 256–9. PubMed Abstract | Publisher Full Text\n\nMolina-Barea R, Rios-Peregrina RM, Slim M, et al.: Lobular breast cancer metastasis to the colon, the appendix and the gallbladder. Breast Care (Basel). 2014; 9(6): 428–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMouchli M, Grider DJ, Yeaton P: Gallbladder Metastases: A Report of Two Cases. Case Rep Oncol. 2019; 12(1): 235–240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiaz N, Ahmed R, Afzal S, et al.: Breast carcinoma with asymptomatic metastasis to the gallbladder. Singapore Med J. 2012; 53(7): e136–8. PubMed Abstract\n\nMarkelov A, Taheri H, Vunnamadala K, et al.: Biliary dyskinesia as a rare presentation of metastatic breast carcinoma of the gallbladder: a case report. Case Rep Pathol. 2011; 2011: 806570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZagouri F, Sergentanis TN, Koulocheri D, et al.: Bilateral synchronous breast carcinomas followed by a metastasis to the gallbladder: a case report. World J Surg Oncol. 2007; 5: 101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbdelilah B, Mohamed O, Yamoul R, et al.: Acute cholecystitis as a rare presentation of metastatic breast carcinoma of the gallbladder: A case report and review of the literature. Pan Afr Med J. 2014; 17: 216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZamkowski M, Kąkol M, Makarewicz W, et al.: Patient with metastatic breast cancer presenting as acute cholecystitis with one-year survival on hormonotherapy. Pol Przegl Chir. 2017; 89(4): 46–49. PubMed Abstract | Publisher Full Text\n\nFleres F, Rossitto M, Foti A, et al.: Metastasis of the gallbladder from the breast cancer. Ann Ital Chir. 2014; 85: pii: S2239253X14023470. PubMed Abstract\n\nHerrera FA Jr, Hassanein AH, Cosman BC, et al.: Breast carcinoma metastatic to the gallbladder and urinary bladder. Eur Rev Med Pharmacol Sci. 2010; 14(10): 883–6 . PubMed Abstract\n\nMachida D, Yukawa N, Gohda M, et al.: A case of metastatic breast carcinoma of the gallbladder. Jpn J Gastroenterol Surg. 2007; 40: 56–62. Publisher Full Text"
}
|
[
{
"id": "64929",
"date": "30 Jul 2020",
"name": "Giorgio Ercolani",
"expertise": [
"Reviewer Expertise :Liver tumor",
"liver 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\nI believe that the case-report is interesting, well reported and should be accepted.\nHowever, I have a minor concern:\nDid the authors perform a liver resection of the gallbladder bed? Is it reported in the literature? Since most of the reported patients died within 2 years from cholecystectomy, the author should discuss if in the finding of incidental metastases from breast cancer, a more extensive procedure (radical cholecystectomy with resection of the gallbladder bed) should be applied similarly to the incindetally gallbladder carcinoma.\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? Partly",
"responses": []
},
{
"id": "72530",
"date": "15 Oct 2020",
"name": "Francesco Fleres",
"expertise": [
"Reviewer Expertise General surgeon",
"Oncologic surgeon",
"colorectal cancer",
"gastric cancer",
"Hepato-biliary-pancreatic surgeon. Hipec",
"Sarcoma",
"Hernia",
"Laparoscopic surgery",
"robotic surgery",
"peritoneal carcinosis."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reported a very interesting and rare case. They have described very well the rarity of the case and oncologic history.\n\nMoreover they have performed an interesting and very complete analysis and review of the literature. It can be accepted as it is.\n\nGood level of language.\n\nIt would be interesting if the authors can describe and argue better on the histologic exam.\n\nI would like to ask some opinions from the authors:\nWhich was the dimension of the metastasis?\n\nWhere was it located, on peritoneal surface or on liver's bed?\n\nHow was the gallbladder extracted? Did you used a endobag?\n\nWas there evident an infiltration of the cistic duct?\n\nOn their opinion and review of literature, should it be considered a more extensive intervention on liver bed as incidental gallbladder cancer?\n\nThe oncologic therapy was performed as hormone therapy and not chemotherapy due the patient's general condition?\n\nThis report and review of literature in very interesting and it can be accepted.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-343
|
https://f1000research.com/articles/7-214/v1
|
22 Feb 18
|
{
"type": "Method Article",
"title": "StateHub-StatePaintR: rapid and reproducible chromatin state evaluation for custom genome annotation",
"authors": [
"Simon G. Coetzee",
"Zachary Ramjan",
"Huy Q. Dinh",
"Benjamin P. Berman",
"Dennis J. Hazelett",
"Simon G. Coetzee",
"Zachary Ramjan",
"Huy Q. Dinh",
"Benjamin P. Berman"
],
"abstract": "Genome annotation is critical to understand the function of disease variants, especially for clinical applications. To meet this need there are segmentations available from public consortia reflecting varying unsupervised approaches to functional annotation based on epigenetics data, but there remains a need for transparent, reproducible, and easily interpreted genomic maps of the functional biology of chromatin. We introduce a new methodological framework for defining a combinatorial epigenomic model of chromatin state on a web database, StateHub. In addition, we created an annotation tool for bioconductor, StatePaintR, which accesses these models and uses them to rapidly (on the order of seconds) produce chromatin state segmentations in standard genome browser formats. Annotations are fully documented with change history and versioning, authorship information, and original source files. StatePaintR calculates ranks for each state from next-gen sequencing peak statistics, facilitating variant prioritization, enrichment testing, and other types of quantitative analysis. StateHub hosts annotation tracks for major public consortia as a resource, and allows users to submit their own alternative models.",
"keywords": [
"epigenomics",
"chromatin",
"visualization",
"methylation",
"variant annotation",
"ChIP-seq",
"bioconductor"
],
"content": "Introduction\n\nChromatin segmentations are increasingly important for a broad area of research that includes regulatory genomics, genetic epidemiology, precision health, and molecular genetics. There is a need for consistent, unbiased resolution of chromatin states to interpret the epigenome and predict function across different tissues and cell types.\n\nComplex, overlapping patterns of post-translational modifications (PTM) to histone subunits1,2, signify differing states of chromatin activity. These modifications consist of mono-, di-, or tri-methylation and acetylation of histone 3 lysines 4, 9, 27, and 363. Direct assays for histone PTMs with next-generation sequencing (NGS) using chromatin immunoprecipitation (ChIP-seq) result in a set of genomic intervals with evidence for enrichment over background (input chromatin), using signal intensity.\n\nIn addition to ChIP-seq of histone PTMs, there are also NGS methods for histone displacement, including DNase I hypersensitivity4 (DNase-seq or DHS), Formaldehyde Assisted Isolation of Regulatory Elements5 (FAIRE-seq), Assay for Transposase Accessible Chromatin6 (ATAC-seq) and Nucleosome Occupancy and Methylome sequencing (NOMe-seq)7. Histone displacement, nucleosome positioning and DNA methylation are also detected in genomewide assays (e.g. whole genome bisulfite sequencing8). Histone displacement is associated with transcription factor binding and transcriptional activity9. In addition, direct binding of transcription factors is measured in ChIP-seq experiments with an antibody directed against a transcription factor or an epitope-tagged version.\n\nAll these data are compatible with data represented as genomic intervals (in bed format), including CpG islands, annotated transcription start sites, repeat elements, 3′ UTRs. The input and final (output) processed data format are both as browser extensible data (.bed), a flexible standard for different peak calling methods (e.g. “narrowPeak” and “broadPeak” are types of .bed files).\n\nSeveral machine-learning approaches integrate NGS experiments into annotation tracks10. The goal is to discover epigenomic states and aid in understanding “non-coding” genomic elements in an unbiased and biologically meaningful way. Newly discovered states are likely an amalgam of true functional categories reflected in chromatin biology. The most popular and widely used of these machine learning methods is ChromHMM11. Other machine-learning approaches include spectral-based learning12, inference based on read counts13, dynamic bayesian networks14, probabilistic approaches15, supervised enhancer detection16, and other hidden Markov methods17–19.\n\nThe interpretability and general usefulness of the state predictions produced by these algorithms varies. A multitude of states often must be consolidated into simpler, biologically meaningful categories. Hoffman et al., recognized this problem when they proposed a combined meta-analysis of ChromHMM and Segway annotations20. However, a software framework for expert or rule-based segmentations is still lacking. Such methodology is needed for integrating different experimental data (including non-NGS data) in a reproducible way, reflecting both the novel insights gained from the machine learning methods and our current understanding of genome biology.\n\nHere we introduce StateHub and StatePaintR for generating and documenting chromatin state and other genome segmentation models in a transparent and reproducible fashion. StateHub is a public resource for storing annotation models, state definitions and associated data in a shareable, referenceable form. The StatePaintR package implements these models and state definitions to produce annotation tracks. We show that StatePaintR can be used to rapidly annotate large collections of public data for summarizing epigenomics data or annotation of variants. We show how annotations gracefully degrade, in that cell types or tissues with missing data types are annotated appropriately based upon available information. We show some use cases, and describe how StatePaintR uses ChIP-seq data peak statistics to rank the state prediction for each segment.\n\n\nMethods\n\nStatePaintR is implemented as a software package in the R language freely available from the Bioconductor repository: www.bioconductor.org/packages/release/bioc/html/StatePaintR.html. The package contains functions for generating annotation tracks according to the rules specified in a model matrix. The matrix cell values can take any of 4 different values (Table 1) representing the relationship between the row labels (proposed state) and column labels (functional categories). The current package only accepts these 4 values, since each value produces very specific behavior from the software.\n\nStatePaintR assigns annotations according to custom rules specified in a matrix. The rules are represented as an integer code that takes any of 4 values [0–3]. The meaning of each value is summarized in the table below.\n\nEach cell of the model matrix relates functional category to chromatin state in a 3 bit code representing the answers to two TRUE/FALSE questions (see Table 1). Is the functional category required in order to call the state? And, is overlap consistent with the state? For example, in our focused poised promoter model, the cell of the decision matrix defining the relationship between the state “PPR” and the functional category “PolycombNarrow” is 3, representing the binary value 112. In order to call the PPR state, PolycombNarrow data is required be present, and second, it must also overlap with a peak. A score of 2 representing the binary value 102, as in the category “Active” and state PPR, indicates that data relating to the active mark must be present to call PPR, but must not overlap. A score of 0 representing the binary value 002, as in the cell for category “Core” and PPR, indicates that it is not necessary for data consistent with Core marks to be present. If the data is present and overlapping a segment, the state is excluded. The category “Translation marks” does not affect PPR in this model, even if it overlaps. Marks that are essentially irrelevant to PPR such as this one are assigned 1 representing binary 012.\n\nThus, each row (as “state”) has a unique combination of matrix values, and the rows are organized by the software in order of increasing information content (as row sums). StatePaintR first generates a GRanges list (an R object containing a list of chromosomes and interval coordinates with arbitrary metadata columns attached) of all uniquely mapping segment boundaries from the start and end coordinates of every peak in all files. StatePaintR then evaluates the presence or absence of each mark and eliminates erroneous states. Next the program assesses overlaps of each segment to determine whether the conditions specified in each cell of the decision matrix are compatible with that segment, producing a boolean value. Rows with perfect matches in all cells are candidate state calls. Since StatePaintR evaluates in order of increasing information content, lower information states can be overwritten if higher information states match. This is very useful for building degeneracy in a model. An example of this in Figure 1 is illustrated by the states ER and EAR. If active marks (e.g. H3K27Ac) are not available for a given cell type, StatePaintR will annotate H3K4me1 marks as ER under our default model. In a different cell type for which H3K27Ac data are available, StatePaintR will know to distinguish between H3K4me1 enriched regions as either active or poised based on overlap of this second mark. Thus, a model can specify different state calls as appropriate based on the availability of data for each cell type. StatePaintR includes a peak score for each state drawn from all experiment categories (columns) that have a matrix value of 3, i.e. because they are required for and consistent with that state. The peak scores are rank normalized on a scale of 1 to 1,000, with 1 being the minimum peak size and 1000 being the maximum. If multiple categories are required, StatePaintRselects the median peak score for the annotation. This behavior can be overridden (see documentation for details). The package includes an R-markdown vignette. The current release version of this vignette is always available from the Bioconductor website.\n\nExperimental data and external database annotations are combined into abstraction layers (columns), integrated to produce chromatin states (rows) from the decision matrix. StatePaintR produces state assignments by iteratively comparing the marks that are present in each segment with each row of data in the table. The values of color-coded squares signify relationship between data and state: 0 (light red) the feature/data type negates the state but is not required to be present, 1 (light green) feature is consistent with the state but not required, 2 (red) if the feature is required to be available and negates the state, and 3 (green) it is both required and consistent with the state. Information content (sum of row values) of states increases from top to bottom. For the example, red dotted arrows indicate non-matching rows, and green arrows indicate matching rows. The state call corresponds to the last matched row.\n\nStateHub is implemented as an interactive website (www.statehub.org). StateHub contains a database implemented in MongoDB and a search engine written with Google Web Toolkit (GWT), which updates dynamically with user input. This database includes all models, model metadata and pre-computed StatePaintR browser tracks. Models are composite JSON objects that include an unique identifier, name, revision number, a searchable text description, and a model matrix (as defined in Table 1). The website also includes links to this manuscript, R-markdown containing code for figures, the latest version of the vignette, links to twitter feed and additional instructional materials.\n\nThe main text makes reference to two models in StateHub (statehub.org). The unique identifiers of these models are as follows: “Default” (model ID: 581ff9f246e0fb06b4b6b178) and “Focused Poised promoter” (model ID: 5813b67f46e0fb06b493ceb0). In each of the two models presented and discussed in this paper we chose a naming convention for our states reflecting biological function.\n\nPreprocessed peak calls were obtained from the IHEC and ENCODE websites (see Table 2) for hg19, and where possible hg38. Where possible we used IDR (Irreproducible discovery rate) processed narrowPeak calls for DHS and broadPeaks for broad marks (H3K27Ac, H3K4me1, H3K27me3, H3K36me3) unless otherwise specified in the model. A complete manifest with filenames, plus all annotation tracks are available on the StateHub website.\n\nData from the indicated public consortia were downloaded and processed in StatePaintR. The resulting annotation files and browser sessions are available from the StateHub web page under each model page.\n\nParkinson’s GWAS variants. To illustrate the use of StatePaintR chromatin state segmentations in GWAS functional annotations, we revisited an earlier study of Parkinson’s disease in which tested for tissue-specific enrichment of genetic associations. Parkinson’s GWAS variants were obtained from a previously published large scale meta-analysis21. We used a beta-binomial conjugate distribution to estimate the credible range of differences in overlaps between observed (GWAS hits) vs. random variants. To calculate enrichment we selected all variants within 1 MB of the index SNP in each region with a minor allele frequency (MAF) > 0.01, defining foreground as SNPs in linkage disequilibrium with the index SNP at a cutoff of r2 > 0.8 and background as all SNPs inclusive (MAF > 0.01). Enrichment in genomic annotations. Analyses and graphics were produced using the SegTools package22.\n\nTo select methylation variants, we analyzed the Infinium HM450 data of 114 ovarian tumor samples23 and 216 control normal Fallopian tube samples24. We define differentially methylated regions as those having a difference in beta values of 0.3 (cancer vs. normal) and significance in Mann-Whitney U-test (FDR-corrected p-value < 0.01). We then performed enrichment calculations using overlaps between probes that were hypermethylated in cancer vs. normal and the state calls from two models described above and in the text. The enrichment calculations were as described in the previous section, treating methylation “variants” analogously to SNPs. We used the complete HM450 probeset as background.\n\nAll code used to generate figures, tables, and this manuscript is included as an R-markdown document (Supplementary File 1). A copy of this document may also be obtained from the StateHub website. In addition, a workflow vignette is available from the bioconductor package and mirrored on the github repository at github.com/Simon-Coetzee/StatePaintR.\n\n\nResults\n\nIn order to assign chromatin states, it is necessary to account for the complex interplay of input from genomic annotations and cell-type-specific experimental data sources that define and demarcate functional regions of the genome1. Computationally they have to be put in the right order to avoid erroneous overwriting of information-rich categories with information-poor ones.\n\nWe initially wrote a model as a decision tree, encompassing a set of basic rules for annotation, but quickly discovered that any small change to the model necessitated a near complete re-write of our software. Secondary to this, we wanted a solution that would enable us to specify any change in the model and have it produced the same way as all previous models while minimizing software updates. And thirdly, we felt that any such model should be reproducible, documented, cite-able and extensible to any combination of experiments. Moreover from a bioinformatics perspective, we felt that any two colleagues working separately should be able to produce precisely the same annotations from the same datasets and models. To satisfy these different requirements we separated the model specification from the annotation tool. We implemented model-specification as a decision matrix, which has the advantage of separating model specification from software, enabling complete explicit control of the annotation software without computer programming expertise.\n\nWe created a searchable website, StateHub, to host a permanent repository of models, document model objects and make them available as a resource to the community. The StatePaintR package retrieves models from StateHub and performs annotations on local data. Thus, StateHub-StatePaintR is a framework to document models and apply them to annotate genomic data. The models in StateHub consist of an abstraction layer, defining the relationships between data sources and functional categories. These categories are integrated to produce annotations (left hand column, “Chromatin States”) via a decision matrix (Figure 1). Within the model each state has associated descriptions of arbitrary length which may contain key words or other relevant details (bottom right).\n\n\nUse Cases\n\nWe generated annotations of 119 ENCODE cell lines25, 128 Roadmap tissues26, 26 cell lines and tissues from CEEHRC (peak calls obtained from the IHEC website), and 23 blood cell types from Blueprint (download at statehub.org). On a desktop PC it takes approximately 12–15 seconds to produce an annotation from a typical cell line, depending on the number of datasets and intervals (see Figure S1). StatePaintR produces genome browser compatible BED files with color-coded state annotations (specified in StateHub model). Figure 2 shows a representative region around the POLR2A gene from a subset of 77 high-quality(at least 15m reads) tissue samples and cell lines with H3K27Ac data from Roadmap. A complete manifest for processing these data is included in additional files 1.\n\nAnnotations of 77 cell types from the Roadmap Epigenomics consortium, including some Roadmap-processed ENCODE data, selected for their high quality with default model. Roadmap tissues are clustered and color coded at left according to the same color scheme used in Roadmap publications26.\n\nA common use of genome annotation is to assign putative function to genetic loci identified by genome-wide association studies (GWAS), particularly for non-coding regions. We previously used a custom annotation of Roadmap tissues based on the approach described in this manuscript to identify locus-specific tissue enrichment in variants associated with Parkinson’s disease27. In that study, we displayed locus-by-tissue enrichment as a heat-map. Here we present a similar analysis using our new StateHub model as the basis for an alternative visualization. Since we showed that Parkinson’s disease variants are primarily associated with enhancers and promoters27, we plotted the 95% range of credible values for enrichment in enhancers and promoters vs background SNPs (matched for GC content & minor allele frequency). Each locus (row) is plotted against a selection of tissues in Roadmap (Figure 3).\n\nBars: 95% credible range for enrichment in active states vs SNPs in the region with similar minor allele frequency and LD < 0.8, for each of 4 independent genetic loci. θ1, θ2 relative enrichment in foreground and background sets, respectively. a1, b1 number of foreground SNPs overlapping biofeatures or not-overlapping, respectively. a2, b2 number of background SNPs overlapping biofeatures or not-overlapping, respectively. a and b are shape parameters of a beta distributed prior. Significant enrichment profiles for roadmap tissues are displayed in color (REMC lineage-specific colors); non-significant are gray.\n\nOur “default” model proposes a class of enhancers and promoters in a poised state (EPR and PPR). These features have H3K4me1 or H3K4me3 and lack H3K27Ac. This model also classifies H3K27me3 as silenced/polycomb repressed (SCR). To investigate functional enrichment of methylation variants, we looked at how differentially methylated regions (DMR) in ovarian cancer tumors partition between chromatin states as defined in this model (Figure 1).\n\nFrom previous work, CpG islands containing temporarily silenced (poised) genes by polycomb repressive complex in normal tissues may acquire DNA methylation during cancer formation resulting in permanent silencing28,24. While the segments called EPR and PPR were associated with hypermethylated probes in ovarian cancer across tissues, the magnitude of enrichment was not great (see Figure 4, “Model 1”), and it remained possible that our state definitions were too broad.\n\nEnrichment as in Figure 3 using either of two different state models (model 1 and model 2) from StateHub, “Default” and “Focused Poised Promoter”, which differ in the treatment of poised promoters. Y-axis range is the same for both plots. Both models distinguish hypermethylated probes in the poised state but model 2 is more selective than model 1. In this model (2) enhancers with H3K4me1 and promoters with H3K4me3 overlapping narrow regions of H3K27me3 are poised (EPR and PPR), but those without H3K27me3 are called weak (EWR and PWR). Model 1, by contrast, assigns promoters lacking active marks to the poised state.\n\nOne hypothesis is that poised promoters are distinguishable by the presence or absence of focused H3K27me3, in particular the narrowPeak calls (as opposed to broad, low-level enrichment from broadPeak files used in model 1). To address this hypothesis, we repeated the analysis in Figure 4 for an alternative model (model 2; “focused poised promoter”) in which H3K27me3 is called as both broadPeak and narrowPeaks. We use the H3K27me3 broadPeak file as in the previous model to identify repressed regions, and H3K27me3 narrowPeaks to identify poised states (EPR and PPR). Enhancers lacking H3K27Ac and H3K27me3 were classified as weak enhancers and promoters (EWR and PWR, not shown in Figure 4). Regulatory elements with these properties have been also been called “primed”29.\n\nWe found greater enrichment when we defined poised states in this way (compare model 2 (focused poised promoter) with model 1 (default) in Figure 4). The hypermethylated ovarian cancer CpGs were more enriched in EPR, PPR, and SCR states as defined in the focused poised promoter model relative to the default model, and hypomethylated probes were enriched only in HET and SCR states (not shown). The odds ratio of enrichment for hypermethylated CpGs in EPR and PPR from the default model fell in a range between 0 and 5. However, the enrichment of the hypermethylated probes in our focused poised promoter model was > 5 in PPR and > 10 in EPR (Figure 4, model 2). Thus, ovarian hypermethylated probes are enriched across roadmap tissues in H3K27me3+ enhancers and promoters, and we concluded that H3K27me3 narrowPeaks are an important distinguishing feature for this class.\n\nNext, we characterized the distribution of states in our focused poised promoter model relative to Gencode v37 gene annotations and also to enhancers from Ensembl30. Figure 5 shows the relative enrichment of Human mammary epithelial cell (HMEC) chromatin states in each of these features. We found enrichment in Ensembl enhancers for three states: Active enhancer (EAR), Active regions (AR) and Weak enhancer (EWR). The definition of “active enhancer” in the Ensembl build is cumulative across cell types30 and therefore includes many cell-type specific enhancers that would be predicted to be weak (having exclusively H3K4me1) in a particular cell line such as HMEC. These three states were not enriched in any other category of genomic annotations. Likewise we found enrichment of the inactive enhancers in Transcribed (TRS) and Silenced/Polycomb (SCR). TRS was most enriched in gene body annotations, particularly internal exons and introns. SCR and Heterochromatin (HET) were depleted across all categories. Lastly, the 5′, first exon and first intron regions were enriched in active and weak promoters, consistent with the role of these regions in transcription initiation.\n\nRelative enrichment of called states genomewide from HMEC in annotations from Ensembl and Gencode. Genegraph (top) visualization of the regions indicated for each column. Enrichment is log2 observed over random. Positive enrichment is indicated with mustard color (scale from 0 to 0.66) vs. relative depletion in purple (scale from 0 to -0.37).\n\nTo use ChIP-seq data for quantitative analysis, we ranked within each state by peak score from Macs2 output (generic peak height). We programmed StatePaintR to rank each state by normalizing on a scale of 1-1000, 1000 being the highest rank. StatePaintR ranks the required dataset(s) for each state (i.e. assigned “3” in the decision matrix). To evaluate the ranking function, we measured area under the precision-recall-gain curve (AUPRG) using the set of experimentally validated human and mouse noncoding fragments with gene enhancer activity as assessed in transgenic mice (VISTA enhancer browser and31). We randomly sampled 100 enhancers from 7 VISTA tissues to evaluate different aspects of our models (training), and then used the remainder of the data to test our enhancer predictions against previously published predictions using the same data sets.\n\nSome states, including the ones that are germane for enhancer prediction, reference more than one required (matrix value 3) dataset, and therefore it was necessary to optimize the best method for ranking based on > 1 ChIP-seq experiment. We computed the average, median and ceiling functions of ranks across multiple ChIP-seq tracks. The three methods were comparable, but median and average produced the best results (Figure S2). There are three required marks for active enhancers in our model, but if one of them is not informative for active enhancer prediction, using the ceiling “max” method would produce false positives when this mark has the highest peak rank. Therefore we interrogated which marks are informative using a leave-one-out approach. We found that leaving out H3K4me1 significantly improved our predictions, whereas leaving out the other marks did not (Figure S3).\n\nNext we assessed AUPRG of different state calls vs. VISTA enhancers and found that predictive power descends in order AR + EAR > EAR > AR > RPS > EPRC > etc (Figure S4). When we tried combinations of states the highest precision recall gain was observed for EAR, EARC, AR and ARC added together (Figure S4), and this was greater than other combinations and than any of the state calls individually. H3K27Ac is the only mark common to all these states, suggesting that H3K27Ac is the most informative predictor of enhancers.\n\nSince H3K4me1 does not improve predictions and is the only thing that distinguishes between AR and EAR (by its presence or absence), an improved model would consolidate AR and EAR into a single state and reassign “1” to H3K4me1 instead of “3”, leaving this mark exclusively to define weak (or primed) promoters.\n\nTo validate our method of enhancer prediction, we compared our predictions with ENCODE Encyclopedia, Version 3 (zlab-annotations.umassmed.edu), EnhancerFinder, RFECS, DELTA, CSIANN, and REPTILE32–36 for held-out data using AUPRG (Figure S5)37.\n\nOur predictions are comparable to the Encode model that uses H3K27Ac overlapping with distal DHS, RFECS and REPTILE, which had the lowest average rank across tissues (Table 3, Figure S5). Our predictions compared favorably to EnhancerFinder and CSIANN which had an average rank > 6 across the different tissues; heart, midbrain, hindbrain, neural tube and limb. Predictions are only available for these tissues. Thus, StatePaintR ranking is useful for drawing quantitative comparisons between different models, making predictions, or prioritizing regions for functional evidence.\n\nColumns 2–6 reflect the area under the precision-recall gain (auprg) curve.\n\n†Annotations using “poised promoter” model as described in the text.\n\n\nDiscussion\n\nWe created a platform for hosting, browsing, and generating new genome annotation models called StateHub. The StateHub framework makes it possible to specify combinations of genomic data as they relate to regions of functional significance in epigenetically marked chromatin. In addition, we created a software package, StatePaintR, that facilitates the use of StateHub models to generate browser tracks for bioinformatic analyses. We showed how StatePaintR can be used as part of a workflow with uniformly processed data to generate reproducible annotations from public and private data.\n\nOur framework does not replace current machine learning methods, the aim of which is to discover states. But these methods suffer from certain drawbacks that we have addressed with a rules-based approach that provides greater transparency and reproducibility. For example, it is often the case with machine-learning methods that more states are discovered than immediately understood, and there have been different solutions proposed. During discovery, one could iteratively reduce the number of states, minimizing the number of similar or redundant combinations of histone marks. Then the number of discovered states would depend on the number of unique data types used for learning and their distribution around known features. This brittleness makes replication in different settings (with different labs or types of experiments) impractical. Our method avoids these issues, allowing users to specify a model of the epigenome in a matrix (as in Figure 1) that accounts for all known possibilities. Thus, we built a comprehensive framework for a rules-based annotation, reflecting current hypotheses (or models) of the epigenome.\n\nA significant drawback of our approach is that some unusual combinations of marks that may have biological function will be ignored. This has much to do with the fact that StatePaintR is not for discovering novel states, but rather for annotating the genome according to a specific, existing model. Nonetheless, the label assignment step of other chromatin state discovery tools also suffers the same limitations; states are aggregated or optimized in an iterative fashion based on prior knowledge and assumptions. ENCODE for example has published tracks for both ChromHMM and Segway that include multiple states with similar names (e.g. “Tss” vs. “TssF” from ChromHMM, and “EnhF1” vs. “EnhF3” from Segway20). To resolve discrepancies between the two methods, the authors of those studies proposed a combined analysis to simplify the number of state labels and summarize discovery using a rule-based metric not unlike a StateHub model. Thus, they classified regions into 7 types “emphasizing biologically meaningful differences”20. In direct comparisons, we found that our own annotations exhibited greater similarity to the combined analysis than to either of the Segway or ChromHMM tracks separately (not shown). Whatever the protocol, the basic problem persists; machine-learning is able to provide insight into what the categories are, but not how many categories there should be. Currently this remains the exclusive province of the biologist.\n\nOne of the additional challenges is compatibility between data sets. In order for two or more cell types to be annotated according to the same model, it is necessary to combine each of the cell types for the training. One solution is concatenation of genomes20. Another approach is to jointly model epigenomes in parallel, as proposed in Integrative and Discriminative Epigenome Annotation System (IDEAS)38. This approach has the distinctive advantage of also modeling segment boundaries. Our approach does not model boundaries, but does offer some advantages. One is reproducibility: StatePaintR always produces the same annotation independently for each cell type from the same model. Secondly, even samples with different types of data or missing data result in compatible annotations because they come from the same model. Third, the models, composed of a 2D matrix with a range of 4 values, are relatively easy to understand and author. Every file produced in StatePaintR contains a record of the model ID, genome version and all the source files. Clinicians working with human genetics will value consistency and reproducibility across datasets. We produced annotations for REMC, ENCODE, IHEC and blueprint and made these available on the StateHub website for the two models described in this paper. The website also has links to browser sessions where they can be explored and used to create figures. A fourth advantage is speed: samples can be processed in parallel and there is no computationally expensive learning step.\n\nA final feature that is very useful is the ranking by peak score (Figure S5). Using this scheme, we investigated what states contribute most to true enhancers (Figure S2–Figure S4). We found that H3K27Ac defined the best predictive subset of annotations for VISTA enhancers. We also investigated different approaches for handling multiple peak calls for a state and found the median to be optimal (Figure S2), and incorporated this method as the default behavior of StatePaintR. When we compared our predictions to held-out data, they were comparable to the best enhancer predictions36,33 and ENCODE enhancers25 and on the web (unpublished).\n\nWe demonstrated a workflow wherein new models generate annotations, which are used to test predictions against experimental data, and then in turn to make improvements to old models. We anticipate that this will be valuable in testing new ideas and hypotheses generated from unsupervised methods. The ability to rank features also aids in prioritizing variants for GWAS and studies of somatic mutations. Knowing which variants overlap features in the epigenomic landscape of a particular cell type is key. In the future, other methods may become available for incorporation into StatePaintR but the models described in StateHub will remain stable.\n\n\nConclusions\n\nWe introduced two new computational resources, an online database of chromatin state models and processed genome segmentations called StateHub, and an R/Bioconductor tool called StatePaintR, which translates epigenomics files into segmentations using these models. One may annotate incomplete datasets rapidly and sensibly according to a single model specification that gracefully degrades to lesser annotations with missing data. Annotations have header documentation with genome version, StateHub model, and the names of source files and their mappings. These tools document segmentations and state labels precisely as they are used in individual studies and to allow comparisons between evolving models of epigenomic states as they relate to NGS experiments. They also enable mixing of epigenomic states with other types of data, such as 3D looping assays, transcription factors, primary sequence features such as position weight matrices, or disease variants.\n\n\nData and software availability\n\nThe following are additional files containing manifests to run StatePaintR with current releases of all public datasets listed in Table 2, links to segmentation tracks, and all code used for analysis and generation figures in this manuscript. Complete code generated from R markdown (Rnotebooks/html format) for generating all analyses, figures and tables is available here.\n\nStateHub available from: http://statehub.org/\n\nArchived source code of StateHub as at time of publication: https://zenodo.org/record/114879239\n\nStatePaintR available from: http://www.bioconductor.org/packages/release/bioc/html/StatePaintR.html\n\nSource code of StatePaintR availabe from: http://www.github.com/Simon-Coetzee/StatePaintR\n\nArchived source code of StatePaintR as at time of publication: https://zenodo.org/record/113782540\n\nLicense: GPL v3.0\n\nAt the time of publication we have submitted our package to Bioconductor. A new version of the article will be updated once this package is available. For now, the entire package is available on GitHub",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe study was funded by National Institutes of Health (UO1CA184826 (BPB) and RO1CA190182 (DJH)).\n\n\nSupplementary material\n\nSupplementary File 1: Statepaintr.nb.html: This file contains code for all the examples and use cases in the text of this manuscript, generated as an html from Rmarkdown. Archived version of this file as at time of publication is available from: https://zenodo.org/record/113782540.\n\nClick here to access the data.\n\nFigure S1: Relationship between data and runtime. StatePaintR takes only a few seconds to run. The exact time depends on the number number of unique segments (lines of data) created by overlapping genomic intervals of all input files, cumulative. Thus, 128 Roadmap tissues can be run in 10 sec × 128 ≈ 1,280 sec (21 min).\n\nClick here to access the data.\n\nFigure S2: Predictions with multiple marks. Ranked ChIP-seq peak scores for multiple marks were used to rank active enhancers (H3K4me1 + H3K27Ac + DHS) by 3 methods (median, mean, ceiling) and compared to a sample (n = 100) of experimentally validated enhancers. The average or median of three marks was a better predictor than ceiling. The choice of function is subservient to choice of data for ranking–if one of the three is less informative, it will produce false positives when using the max method–therefore it is better to eliminate uninformative marks. See also Figure S4.\n\nClick here to access the data\n\nFigure S3: Ranking enhancers with subsets of marks. Combinations of marks were used to predict active enhancers by the max ranking method (as in Figure S2) and compared to enhancer score. “All” includes regulatory (H3K4me1), active (H3K27Ac), and core (DHS). We also tried a leave-one-out strategy for each of these categories in succession. Leaving out H3K4me1 (“no regulatory”) produced superior predictions, suggesting that its inclusion made the predictions less specific.\n\nClick here to access the data\n\nFigure S4: Chromatin states as predictors of true enhancers. We tested different chromatin states for their ability to predict true enhancers under the poised focused promoter model. Active enhancers exhibited the greatest predictive power under the precision recall gain curve.\n\nClick here to access the data\n\nFigure S5: Performance of enhancer predictions. Area under precision-recall-gain curves reflect the accuracy of three models of enhancer prediction. True positive enhancers are those validated in the VISTA enhancer browser. The ENCODE method (in blue) and the StatePaintR method (in red) show similar accuracy in retrieving VISTA enhancers showing tissue specific enhancer activity, while EnhancerFinder (in green) is less accurate.\n\nClick here to access the data\n\n\nReferences\n\nRando OJ: Combinatorial complexity in chromatin structure and function: revisiting the histone code. Curr Opin Genet Dev. 2012; 22(2): 148–155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardner KE, Allie CD, Strahl BD: Operating on chromatin, a colorful language where context matters. J Mol Biol. 2011; 409(1): 36–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothbart SE, Strahl BD: Interpreting the language of histone and DNA modifications. Biochim Biophys Acta. 2014; 1839(8): 627–643. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoyle AP, Davis S, Shulha HP, et al.: High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008; 132(2): 311–322. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon JM, Giresi PG, Davis IJ, et al.: Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nat Protoc. 2012; 7(2): 256–267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuenrostro JD, Giresi PG, Zaba LC, et al.: Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013; 10(12): 1213–1218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGal-Yam EN, Jeong S, Tanay A, et al.: Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting. PLoS Genet. 2006; 2(9); e160. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCokus SJ, Feng S, Zhang X, et al.: Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature. 2008; 452(7184): 215–219. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThurman RE, Rynes E, Humbert R, et al.: The accessible chromatin landscape of the human genome. Nature. 2012; 489(7414): 75–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Y, Chen CY, Kaye AM, et al.: The identification of cis-regulatory elements: A review from a machine learning perspective. Biosystems. 2015; 138: 6–17. PubMed Abstract | Publisher Full Text\n\nErnst J, Kellis M: ChromHMM: automating chromatin-state discovery and characterization. Nat Methods. 2012; 9(3): 215–216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong J, Chen KC: Spectacle: fast chromatin state annotation using spectral learning. Genome Biol. 2015; 16(1): 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMammana A, Chung HR: Chromatin segmentation based on a probabilistic model for read counts explains a large portion of the epigenome. Genome Biol. 2015; 16(1): 151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffman MM, Buske OJ, Wang J, et al.: Unsupervised pattern discovery in human chromatin structure through genomic segmentation. Nat Methods. 2012; 9(5): 473–476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHon G, Ren B, Wang W: ChromaSig: a probabilistic approach to finding common chromatin signatures in the human genome. PLoS Comput Biol. 2008; 4(10): e1000201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantoni FA: EMdeCODE: a novel algorithm capable of reading words of epigenetic code to predict enhancers and retroviral integration sites and to identify h3r2me1 as a distinctive mark of coding versus non-coding genes. Nucleic Acids Res. 2013; 41(3): e48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZacher B, Lidschreiber M, Cramer P, et al.: Annotation of genomics data using bidirectional hidden Markov models unveils variations in Pol II transcription cycle. Mol Syst Biol. 2014; 10(12): 768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSohn KA, Ho JW, Djordjevic D, et al.: hiHMM: Bayesian non-parametric joint inference of chromatin state maps. Bioinformatics. 2015; 31(13): 2066–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiesinger J, Wang J, Xie X: Discovering and mapping chromatin states using a tree hidden markov model. BMC Bioinformatics. 2013; 14 Suppl 5: S4. PubMed Abstract | Free Full Text\n\nHoffman MM, Ernst J, Wilder SP, et al.: Integrative annotation of chromatin elements from ENCODE data. Nucleic Acids Res. 2013; 41(2): 827–841. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNalls MA, Pankratz N, Lill CM, et al.: Large-scale meta-analysis of genome-wide association data identifies six new risk loci for Parkinson’s disease. Nat Genet. 2014; 46(9): 989–993. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuske OJ, Hoffman MM, Ponts N, et al.: Exploratory analysis of genomic segmentations with segtools. BMC Bioinformatics. 2011; 12: 415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatch AM, Christie EL, Etemadmoghadam D, et al.: Whole-genome characterization of chemoresistant ovarian cancer. Nature. 2015; 521(7553): 489–494. PubMed Abstract | Publisher Full Text\n\nTeschendorff AE, Gao Y, Jones A, et al.: DNA methylation outliers in normal breast tissue identify field defects that are enriched in cancer. Nat Commun. 2016; 7: 10478. PubMed Abstract | Publisher Full Text | Free Full Text\n\nENCODE 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\nThe Roadmap Epigenomics Consortium, Kundaje A, Meuleman W, et al.: Integrative analysis of 111 reference human epigenomes. Nature. 2015; 518(7539): 317–330. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoetzee SG, Pierce S, Brundin P, et al.: Enrichment of risk SNPs in regulatory regions implicate diverse tissues in Parkinson’s disease etiology. Sci Rep. 2016; 6: 30509. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGal-Yam EN, Egger G, Iniguez L, et al.: Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line. Proc Natl Acad Sci U S A. 2008; 105(35): 12979–12984. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalo E, Wysocka J: Modification of enhancer chromatin: what, how, and why? Mol Cell. 2013; 49(5): 825–837. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZerbino DR, Wilder SP, Johnson N, et al.: The ensembl regulatory build. Genome Biol. 2015; 16: 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVisel A, Minovitsky S, Dubchak I, et al.: VISTA Enhancer Browser--a database of tissue-specific human enhancers. Nucleic Acids Res. 2007; 35(Database issue): D88–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErwin GD, Oksenberg N, Truty RM, et al.: Integrating diverse datasets improves developmental enhancer prediction. PLoS Comput Biol. 2014; 10(6): e1003677. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajagopal N, Xie W, Li Y, et al.: RFECS: a random-forest based algorithm for enhancer identification from chromatin state. PLoS Comput Biol. 2013; 9(3): e1002968. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu Y, Qu W, Shan G, et al.: DELTA: A Distal Enhancer Locating Tool Based on AdaBoost Algorithm and Shape Features of Chromatin Modifications. PLoS One. 2015; 10(6): e0130622. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFirpi HA, Ucar D, Tan K: Discover regulatory DNA elements using chromatin signatures and artificial neural network. Bioinformatics. 2010; 26(13): 1579–1586. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe Y, Gorkin DU, Dickel DE, et al.: Improved regulatory element prediction based on tissue-specific local epigenomic signatures. Proc Natl Acad Sci U S A. 2017; 114(9): E1633–E1640. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlach P, Kull M: Precision-recall-gain curves: Pr analysis done right. In: Cortes C, Lawrence ND, Lee DD, Sugiyama M, and Garnett R, editors, Advances in Neural Information Processing Systems. Curran Associates, Inc., 2015; 28: 838–846. Reference Source\n\nZhang Y, An L, Yue F, et al.: Jointly characterizing epigenetic dynamics across multiple human cell types. Nucleic Acids Res. 2016; 44(14): 6721–6731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamjan Z, Coetzee S: zackramjan/statehubweb: initial release of the statehub web frontend app with doi (Version v1.1). Zenodo. 2018. Data Source\n\nCoetzee S: Simon-Coetzee/StatePaintR v0.99.6 (Version v0.99.6). Zenodo. 2018. Data Source"
}
|
[
{
"id": "33280",
"date": "30 Apr 2018",
"name": "Maxwell W. Libbrecht",
"expertise": [
"Reviewer Expertise computational genomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a method for annotating the genome using genomics data sets such as histone modifications, transcription factor binding and methylation. The algorithm is applied to data from a given tissue. It takes as input a collection of genomics data sets that have been binarized in a preprocessing step, such that each is represented by a binary vector over the genome. The method outputs a genomic vector of one of K states, such as \"Promoter\" or \"Transcribed\" (K=20 in their default model). The method uses a \"model matrix\" which defines, for each state-dataset pair, for a given base to be called as that state, if (1) the dataset *may* be positive for that base, and (2) if that dataset *must* be positive for that base.\nStatePaintR is likely to be an impactful method. Genome annotations are a very useful product of epigenomics data sets, as evidenced by the wide array of methods developed for their production. StatePaintR is an alternative to existing algorithms based on probabilistic models that is much simpler and more transparent.\nUnfortunately, the manuscript is difficult to understand in its current form because many key definitions are missing. Several examples: - The term \"functional category\" is not defined. - The Introduction uses the term \"functional category\" to mean a state, where later that term is used to refer to a collection of data sets (such as \"silencing marks\") - The form of the input and output are not explicitly mentioned. - It is not explicitly mentioned that the model matrix is generated manually.\nMinor notes: - It is claimed that the information content of a state equals the sum of the cell values. However, it seems to me that the maximally-permissive value is 1 (neither required nor exclusionary), not 0. - P3: \"3 bit code\". Should be 2 bit code. - Figure 1: \"red dotted arrows indicate non-matching rows\". I don't understand -- each arrow connects to two rows, not just one.\n\nIs the rationale for developing the new method (or application) clearly explained? No\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": "5366",
"date": "07 May 2020",
"name": "Dennis Hazelett",
"role": "Author Response",
"response": "1) First, I would like to thank the reviewer for their comments and corrections. I address them below. “- The term \"functional category\" is not defined. - The Introduction uses the term \"functional category\" to mean a state, where later that term is used to refer to a collection of data sets (such as \"silencing marks\")” The definition of “functional category” has been expanded and clarified in the Implementation section of the methods. Briefly, specific assays may be assigned functional categories via an abstraction layer implemented in StateHub/StatePaintR, where assays that may represent similar biology e.g. ChIP-Seq for H3K27ac and H3K9ac may both be represented by the functional category “Active”. These functional categories may be combined into states following the rules of the decision matrix. “- The form of the input and output are not explicitly mentioned.” The form of the input and output data are now indicated in the Implementation section as consisting of BED files. “- It is not explicitly mentioned that the model matrix is generated manually.” In the expanded explanation of the decision matrix, it has been made clear how they may be constructed manually, or retrieved from StateHub. “- It is claimed that the information content of a state equals the sum of the cell values. However, it seems to me that the maximally-permissive value is 1 (neither required nor exclusionary), not 0.” The reviewer is correct, and the language around this concept has been updated throughout the document. No longer do we refer to information content, as this was an unintentionally misleading term used to describe the complexity of the state. Revised language indicates that potential states are organized by state complexity, with lower complexity states called first. This takes into account the reviewers correct understanding that 1 is the most permissive state. “- P3: \"3 bit code\". Should be 2 bit code.” This correct and has been fixed in the text. “- Figure 1: \"red dotted arrows indicate non-matching rows\". I don't understand -- each arrow connects to two rows, not just one.” The figure legend has been updated to reflect the reading of the figure as a series of arrows, proceeding downward, pointing to the subsequent state. Either red, indicating that the segment was checked as being consistent with the state, and failing, or green indicating that the segment was consistent with the state call. In the context of the figure: “In this example with the presence of H3K4me1 (“Regulatory”), H3K27ac (“Active”) and DNase1 hypersensitivity (“Core”), the first state consistent with the presence of these functional categories is “Enhancer”, followed by the increasingly more complex “Regulatory Site”, “Active Chromatin”, “Active Enhancer”, “Enhancer Core”, “Active Chromatin Core”, and finally “Active Enhancer Core”.”"
}
]
},
{
"id": "33619",
"date": "10 May 2018",
"name": "Yongjin Park",
"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 the paper could be quite impactful and the software they developed can be highly usable. But the paper doesn’t read well. I assume the authors intended to write a research paper, not a technical note. All of my comments are based on this assumption.\nThe authors need to put more efforts to convince ordinary users that StatePaintR is more powerful compared to a single model trained on relevant cell / tissue types. Perhaps expanding from the results in the supplementary section could improve the paper. Why not just train chromHMM or segway given current chip-seq tracks? What’s a clear advantage of the rule-based method? I don' think the rule-based method can clearly estimate underlying model complexity of epigenomics. I think this is too important information to be omitted:\nIn direct comparisons, we found that our own annotations exhibited greater similarity to the combined analysis than to either of the Segway or ChromHMM tracks separately (not shown). Whatever the protocol, the basic problem persists; machine-learning is able to provide insight into what the categories are, but not how many categories there should be. Currently this remains the exclusive province of the biologist.\nDoes this method help prioritize relevant cell / tissue types? Description in the method section is fuzzy. I think a complete paper needs to be self-contained without looking up definitions and terminology from other sources. However, many terms are either vaguely used or never defined. Moreover, the method section needs to be better organized in a top-down fashion instead of enumerating what were implemented. Figure 1 is confusing and not so informative. Why don’t you include real-world example such as chip-seq or methylation tracks? Perhaps you can combine Table 1 with Figure 1. First of all, is Table 1 really necessary? Why do you need both binary and decimal code (I know why but it is irrelevant to the main story of this paper)? It is probably better to show graphical examples how you assign decimal values. How do you define information content? How do you define enrichment? How do you calibrate significance? Is Beta-Binomial reasonable assumption? There are more examples in the background. Do you estimate Beta-Binomial by moment-matching of posterior distribution or maximum-likelihood? y-axis labels are either missing or badly named (Fig 3 and 4). As future direction, how easy is it to implement user-defined enrichment models / methods?\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": [
{
"c_id": "5367",
"date": "07 May 2020",
"name": "Dennis Hazelett",
"role": "Author Response",
"response": "First, I would like to thank the reviewer for their comments and corrections. I address them below. And in the resubmission of the article. “The authors need to put more efforts to convince ordinary users that StatePaintR is more powerful compared to a single model trained on relevant cell / tissue types. Perhaps expanding from the results in the supplementary section could improve the paper.” It is difficult to justify a particular segmentation over another. We propose, rather, that the model espoused by StateHub/StatePaintR of rule-based annotation of predicted states provides an alternative to the existing model of learning the states within the relevant cell/tissue types. The priority of the method is to provide a framework to express existing statements about the relationships of genomic annotations and how they combine to reveal underlying chromatin states thereby bypassing denovo learning and annotating of states within each sample and annotating solely based upon simple rules and available data. However, in order to demonstrate the utility of the method in annotating chromatin states, we have expanded the description of the method’s annotation scoring and how we may predict experimentally validated enhancer regions from the VISTA database. “Why not just train chromHMM or segway given current chip-seq tracks? What’s a clear advantage of the rule-based method? I don' think the rule-based method can clearly estimate underlying model complexity of epigenomics. I think this is too important information to be omitted.” It is true that StatePaintR does not discover new states, nor provide a complete model of the underlying epigenomics. What the tool provides is a language to encode existing knowledge of the relationships between genomic annotations of any kind and the underlying epigenomic state, in a manner that is descriptive, robust to missing data, and consistent across diverse data sets. “Does this method help prioritize relevant cell / tissue types?” This method allows the user to annotate diverse cell / tissue types with a single model. This may provide useful segmentations for the purpose of describing the chromatin state relative to external data, but the method itself does not prioritize specific samples. “Description in the method section is fuzzy. I think a complete paper needs to be self-contained without looking up definitions and terminology from other sources. However, many terms are either vaguely used or never defined. Moreover, the method section needs to be better organized in a top-down fashion instead of enumerating what were implemented.” We agree that the methods were confusing, and have been reorganized and expanded to be more effectively self-contained, clear, and precise. “Figure 1 is confusing and not so informative. Why don’t you include real-world example such as chip-seq or methylation tracks?” Figure 1 is intended to be a schematic representation of how the decision matrix and abstraction layer work together to produce an annotation for a genomic segment. To this end, we have clarified and expanded, both in the figure legend, and the Implementation section of the Methods, the nature of the decision matrix and abstraction layer, including concrete examples of how different chromatin marks combine to form a state. “Perhaps you can combine Table 1 with Figure 1. First of all, is Table 1 really necessary? Why do you need both binary and decimal code (I know why but it is irrelevant to the main story of this paper)? It is probably better to show graphical examples how you assign decimal values.” While the decimal values lend familiarity to the visualization, by defining the two bits that represent the two questions that are answered in the matrix for each cell, we believe that it streamlines understanding of the decision matrix, and also how it works from an implementation standpoint. “How do you define information content? How do you define enrichment? How do you calibrate significance? Is Beta-Binomial reasonable assumption? There are more examples in the background. Do you estimate Beta-Binomial by moment-matching of posterior distribution or maximum-likelihood?” Information content was an unintentionally misleading term that we used to refer to state complexity, so the term information content has been removed from discussion of the decision matrix. For calculating the enrichment within genomic states for SNPs or hypermethylated CpG for figures 3 and 4, we calculate enrichment relative to the appropriate background. As mentioned in Methods: Enrichment Calculations, we considered the background rate of SNPs within active chromatin states to be the proportion of all SNPs within a 1 Mb region of the index SNP with a MAF of greater than 0.01 in the population of interest (Europeans, from 1000 genomes), while the foreground is those SNPs with a linkage disequilibrium R^2 > 0.8. For the methylation data, the full HM450 methylation array was considered as the background, while probes on the array with a difference in beta value between cancer and normal of 0.3 and significance in Mann-Whitney U-test at a p-value of < 0.01. For the GWAS data set, we determined the difference between foreground and background by simulation of posterior draws from the two beta distributions. Significance is determined if the credible interval (95%) did not contain 0. We believe that beta-binomial is a reasonable assumption given that we are controlling the background to be within the exact same genomic region from which we are drawing the foreground, thereby accounting for the heterogeneity of the chromatin states across the genome. For the methlyation dataset, the odds ratio and the 95% confidence interval where calculated with Fisher's exact test. “y-axis labels are either missing or badly named (Fig 3 and 4).” We have corrected the poorly labeled and unlabeled y-axis labels in these figures. “As future direction, how easy is it to implement user-defined enrichment models / methods?” It is not currently easy, though it is possible for users to implement a decision matrix and abstraction layer. The difficulty lies in determining if a model is complete, and non redundant. As a future direction, we intend to create a tool on the StateHub website where models may be submitted and checked for validity, and then, optionally, published for others to use."
}
]
},
{
"id": "33702",
"date": "23 May 2018",
"name": "Guillaume J. Filion",
"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 authors present a pair of tools called StateHub and StatePaintR to annotate genomic states based on chromatin data (ChIP-seq, ATAC-seq etc.). This work has a very pragmatic take on the problem: the software is fast, based on universal rules, linked to a wealth of data etc. For this, the authors have decided to use a rule-based method instead of the traditional machine-learning approach, which in my opinion is completely justified. The early to discover and annotate chromatin states were based on different methods, all “optimal” in their own way. However, none of these methods has proved to have a decisive advantage over the others for the following two reasons: First, chromatin states do not “exist”, they are useful representations associated with a particular state of our knowledge and a particular problem at hand. Second, the choice of the input data and the number of states (i.e. the granularity of the segmentation) seems to be the most influential factor on the end result. With these elements in mind, it makes perfect sense to develop a tool aiming to satisfy the needs of the user and the demand for reproducibility and traceability, rather than some mathematical constraint.\nOverall, the manuscript is well constructed – and as mentioned above it describes a relevant advance – but it could be streamlined for clarity. Many terms are ambiguous (like “active states”) or are jargon for chromatin specialist (like “PolycombNarrow”). The figure legends are barely enough to understand what is plotted and the axes are not all properly labelled. It is a good thing that the authors give some examples to explain the entries of the design matrix. For didactic purposes, they could give more of those, or make the examples more concrete throughout the manuscript to help the reader understand the logic of their tool.\nThe manuscript does otherwise a great job at making the work reproducible, explaining the limitations and the scope of their software, and also at giving a high level description of the implementation. To help the authors sharpen the manuscript for more readability, below is a list of typos and minor issues.\nPage 3, paragraph starting with “All these data...”: Perhaps a word is missing in the sentence “The input and output (final) data are both [?] as browser extensible data…”.\nPage 3, last sentence of the main text: it should read “... PolycombNarrow data is required [to] be present”.\nPage 4, second paragraph, fourth sentence from the end: a space seems to be missing in “StatePaintR[space]selects”.\nPage 4, third paragraph, fourth sentence: “an unique” should be “a unique”.\nPage 4, paragraph “Enrichment calculations”, first sentence. A word seems to be missing in “... an earlier study of Parkinson’s disease in which [?] tested for…”.\nPage 6, paragraph “A framework for rules-based annotation”: “rules-based” should be “rule-based”. See https://english.stackexchange.com/q/1366/44109\nPage 6, paragraph “Segmentation of public datasets”, second sentence from the end, a space is missing before the parentheses in “...high-quality[space](at least 15m reads”. Also, the “m” probably stands for “million” but in scientific texts it must stand for “metres”. If the authors mean “million”, the best option is to write “million”.\nFigure 3, legend: what are “active states”? The authors could give the complete list.\nFigure 4, legend: the authors should indicate on the graph what is plotted on the Y axis (and give the unit). Are the data also plotted in “active” states? Whatever the answer, this should be stated clearly.\nPage 8, first paragraph of the main text, last sentence: there is one “been” too much in “... with these properties have been also been called…”.\nPage 9, first paragraph, last sentence: “roadmap” should be written with a capital R.\nPage 11, second paragraph, second sentence: “rules-based” should be “rule-based” (and again in the last sentence of the paragraph).\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5368",
"date": "07 May 2020",
"name": "Dennis Hazelett",
"role": "Author Response",
"response": "First, I would like to thank the reviewer for their comments and corrections. I address them below. And in the resubmission of the article. “Overall, the manuscript is well constructed – and as mentioned above it describes a relevant advance – but it could be streamlined for clarity. Many terms are ambiguous (like “active states”) or are jargon for chromatin specialist (like “PolycombNarrow”). The figure legends are barely enough to understand what is plotted and the axes are not all properly labelled. It is a good thing that the authors give some examples to explain the entries of the design matrix. For didactic purposes, they could give more of those, or make the examples more concrete throughout the manuscript to help the reader understand the logic of their tool.” The reviewer is correct that legends and the decision matrix were sparsely explained, and the explanation of the decision matrix has been made more clear and explicit, and grounded in an concrete set of examples. Also, figure legends have been expanded, and axes have been made clear. “The manuscript does otherwise a great job at making the work reproducible, explaining the limitations and the scope of their software, and also at giving a high level description of the implementation. To help the authors sharpen the manuscript for more readability, below is a list of typos and minor issues.” Thank you for the corrections, all issues have been corrected, and implemented in the figure legends."
}
]
}
] | 1
|
https://f1000research.com/articles/7-214
|
https://f1000research.com/articles/8-2028/v1
|
29 Nov 19
|
{
"type": "Research Article",
"title": "Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs",
"authors": [
"Sri Suparwitri",
"Paramita Noviasari",
"Paramita Noviasari"
],
"abstract": "Background: Orthodontic tooth movement occurs due to continuous pressure on the teeth, causing the remodeling of the alveolar bone. The tissue will respond to bone growth factors, such as transforming growth factor β1 (TGF-β1), fibroblasts, and bone morphogenetic proteins, for new bone formation. The remodeling process is balanced in young adults, but there is an imbalance in older individuals due to decreased differentiation, activity, and life span of osteoblasts, and increased osteoclasts. Olive oil contains lots of antioxidants and can slow down the aging process. This study aims to study the differences in TGF-β1 levels between old and young guinea pigs, and the difference between olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. Methods: 12 guinea pigs divided into 4 groups: young guinea pigs (4-5 months) not given olive oil; young guinea pigs given olive oil; old guinea pigs (30-31 months) given olive oil; old guinea pigs given olive oil. The teeth were moved using an open coil spring mounted on the brackets on both lower incisors. Gingival sulcus fluid samples were taken on days 0, 7 and 14 of the movement of the teeth. TGF-β1 levels were analyzed using ELISA. Results: Three-way ANOVA and post hoc statistical tests showed that TGF-β1 levels in young guinea pigs were significantly higher than old guinea pigs on days 0, 7 and 14 (p<0.05). TGF-β1 levels in both young and old guinea pigs who were given olive oil was significantly higher than those not given olive oil on days 0, 7 and 14 (p<0.05). Conclusions: TGF-β1 levels in the younger age guinea pigs were higher than the older age, and olive oil could increase TGF-β1 levels in the older age guinea pigs.",
"keywords": [
"olive oil",
"transforming growth factor β",
"orthodontic tooth movement"
],
"content": "Introduction\n\nOrthodontic treatment aims to correct dental malposition, malocclusion, and malrelation of the jaw. Orthodontic tooth movement is a result of the remodeling of periodontal ligaments and alveolar bone1. Bone remodeling during orthodontic tooth movement is described as a continuous and balanced process2. The process of bone remodeling is regulated by osteoblasts and osteoclasts3. Osteoclasts in pressure areas will resorb the alveolar bone so that tooth movement occurs, and this phenomenon will be balanced by osteoblasts in the area4. Transforming growth factor-β1 (TGF-β1) is known as a multifunctional cytokine that plays an important role in bone formation. Moreover, TGF-β1 activity increases with bone formation5.\n\nThe need for orthodontic treatment in adulthood increases with age, but the balance of bone resorption and remodeling processes decreases with increasing age6. Therefore, cellular activity in periodontal tissues of young and old individuals needs to be further studied. This may greatly influence the success of orthodontic treatment7. The process of aging is caused by free radicals, hormones, genetics, lifestyle, diet, pollution, and socioeconomic conditions. If these causative factors can be avoided, then the aging process can be prevented, slowed down, and may even be inhibited8.\n\nOlive oil is extracted from fresh olives that contains many antioxidants and has been shown to slow the aging process9. According to Liu et al., olive oil acts effectively as a substitute for estrogen to prevent bone loss10. Therefore, olive oil is expected to reduce osteoclastogenesis so as to reduce tooth movement orthodontics, especially for cases to prevent loss of anchorage, periodontitis and tooth retention. The purpose of this study is to study the differences in TGF-β1 levels between old and young ages, using guinea pig models, and the effect of olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. The guinea pig was chosen as an animal model as it has been previously used to study the effect of medications on orthodontic tooth movement and has the potential to simulate the response of human tissue11.\n\n\nMethods\n\nEthical clearance was approved by the Research Ethics Committee of the Faculty of Dentistry, University Gadjah Mada (UGM), Yogyakarta, Indonesia (00144/KKEP/FKG-UGM/EC/2019). “Animal Research: Reporting of in vivo Experiments” (ARRIVE) was used for reporting this study. During orthodontic appliance installation and gingival crevicular fluid collection, all the animals were anesthetized. We also used non-invasive techniques to move the teeth (using minimal 35 gr force, which is acceptable for guinea pig), and housing conditions that provided a comfortable and safe environment. All these procedures were done to ameliorate any suffering of animals used in this study.\n\nWe used 12 male guinea pigs (Cavia porcellus) (purchased from a commercial supplier, Kaliurang, Yogyakarta, Indonesia), which were chosen utilizing simple random sampling into four groups (n=3/group): young guinea pigs that were not administered olive oil (young guinea pig-controls); young guinea pigs that were administered olive oil; old guinea pigs that were not administered olive oil (guinea old-control pig); old guinea pigs administered olive oil. Young guinea pigs were 4–5 months old and weighed approximately 300–400 grams, whilst old guinea pigs were 30–31 months old and weighed approximately 800–1000 grams. Sample size for the groups (n=3) was obtained utilizing Lemeshow's formula12. A sample size of three animals in each group would give more than 80% power to detect significant differences with 0.45 effect size and at a significance level of α = 0.05.\n\nAll the animals were maintained for a week before the experiment began on an individual polycarbonate cage (30 cm × 20 cm × 20 cm) with a 12-h light/dark cycle at a steady temperature of 25°C and humidity of 50% for acclimatization to compensate for their various origins. All animals were fed a standard pellet diet (expanded pellets; Stepfield, UK) with tap water ad libitum.\n\nWe used extra virgin olive oil (Bertolli®, USA). Olive oil consumed by humans per day is 30-50 grams so the conversion of a human dose (70 kg) to guinea pig (400 grams) is 0.031. The dose was converted from human olive oil dose (weight, 70 kg) to a guinea pig dose (weight, 400g): 0.031 × 30 ml = 0.93 ml12. Based on body weight, the dose of olive oil given to young guinea pigs (300–400 g) was 0.7 ml and for old guinea pigs (800–1000 g) was 1.86 ml. Olive oil treatment groups (see below) were given olive oil daily from the beginning of the installation of orthodontic devices until day 14. Administration of olive oil was carried out orally with injection syringes that have been cut off (Figure 1) and were given at 9am every morning.\n\nGuinea pigs were first anesthetized with ketamine (160095, Kepro™, Netherlands; 35mg/kgBW) and xylazine (160096, Xyla™, Netherlands; 5mg/kgBW) intramuscularly on the thigh, then a separator was placed between the two lower incisors (Figure 2). Next, the teeth were cleaned with rubber cups and pumice (22257, Kerr, USA) and a Roth bracket 0.022” (American Orthodontics, USA) was installed. The wire arch mounted was 0.018” stainless steel wire arch, which was ligated using power O (American orthodontics, USA). Open coil spring NiTi (American Orthodontics, USA) was installed between the two brackets to move the teeth distally with a force of 0.35 N (Figure 3). The magnitude of the open coil spring force was measured using a tension gauge. The length of the stainless-steel wire was cut 4 mm longer than the open coil spring as a tolerance for orthodontic tooth movement.\n\nGingival crevicular fluid (GCF) collection was performed on days 0, 7, and 14 after olive oil administration. Before removing GCF, the lower incisors of the guinea pig were cleaned with a cotton swab to remove supragingival plaque. Then the teeth were isolated with cotton wool and dried. Next, a 15-point paper point was inserted into the gingival sulcus of the mandibular incisor about 1 mm deep for 30 seconds at an interval of 90 seconds to increase the volume of GCF fluid (Figure 3). The paper point was then inserted into a 1.5 ml eppendorf tube containing 350 μl of saline solution. The eppendorf tube was centrifuged for 5 minutes at a speed of 2000 rpm to elucidate complete GCF components. Paper points were removed and the supernatant solution was stored at -80°C until required for analysis.\n\nDetection of TGF-β1 levels was carried out at the Molecular Biology Laboratory of the Faculty of Medicine UGM using ELISA (enzyme-linked immunosorbent assay). The GCF was analyzed for TGF-β1 levels using ELISA kit reagent test (Cusabio, China), according to manufacturer protocols.\n\nThree-way analysis of variance followed by post hoc statistical tests was used to analyse the difference among the groups and determine its significance. P-values <0.05 were considered statistically significant. All of the data were analyzed using SPSS software (Statistical Package for the Social Sciences, v22.0, USA).\n\n\nResults\n\nAll the experimental procedures were well-tolerated. Furthermore, olive oil administration at the used dosage did not induce any general toxicity, including edema. The results of measurements of TGF-β1 levels of GCF before and after tooth movement can be seen in Table 1. The mean TGF-β1 levels in the older age groups on days 0, 7 and 14 were higher than in the younger age groups, both in the olive oil groups and control groups. The mean TGF-β1 levels in the older and younger age groups increased on day 7 and decreased on day 14 (Table 1).\n\nFigure 4 shows the administration of olive oil increases the average level of TGF-β1 in the young and old age groups. The mean TGF-β1 levels in the olive oil group were higher than the mean TGF-β1 in the group that were not given olive oil (control). The mean TGF-β1 levels in the old control and treatment groups and were higher than the young control and treatment on all days. Based on the time of observation, the highest TGF-β1 level was seen on day 7 and lowest on day 0 in all groups. The highest TGF-β1 level was found in the old guinea pig group treated with olive oil at day 7 and the lowest TGF-β1 level was found in the young guinea pig group who were not given olive oil (young control) on day 0.\n\nNormality test (Saphiro-Wilk) and homogeneity test (Levene) showed normally distributed and homogeneous data. The research data were then analyzed using three-way ANOVA (Analysis of variance). The results of the three-way ANOVA (Table 2) showed that age, olive oil administration and observation time had a significant effect on TGF-β1 levels (p <0.05). There was an interaction between administration of olive oil and observation time, and also between observation time and age, but there was no interaction between administration of olive oil and age or administration of olive oil, observation time, and age (p> 0.05).\n\nA post hoc test was performed after ANOVA to see groups that had significant differences (p <0.05). The post hoc test results showed that most of the TGF-β1 activity of the old and young age groups who were given olive oil and not given olive oil on days 0, 7, and 14 showed significant differences (Table 3).\n\n* = significantly different p<0.05; OC = old, control; OO = Old, olive oil; YC = Young, control; YO = Young, olive oil.\n\n\nDiscussion\n\nThe process of bone remodeling is regulated by osteoblasts and osteoclasts3. Osteoblasts and osteoclasts will appear 40–48 hours after being exposed with orthodontic forces13. Osteoclasts in depressed areas will absorb alveolar bone so that tooth movement occurs. This phenomenon will be offset by the apposition of osteoblasts in the same area4. The balance of bone resorption and remodeling processes decreases with increasing age, which greatly influences the success of orthodontic treatment6,7. Olive oil has been shown to contains antioxidant compounds, namely phenolic compounds. Phenolic compounds are able to provide a strong protective effect against aging14–16. The results of the current data analysis showed that the average TGF-β1 level in GCF in the olive oil groups was higher than the mean TGF-β1 in the groups that was not given olive oil (controls). The results of the three-way ANOVA (Table 2) showed that age, olive oil administration and observation time had a significant effect on TGF-β1 levels (p<0.05). TGF-β1 is a multifunctional cytokine that modulates proliferation, growth, differentiation, adhesion and cell survival, but it also plays a role in the production of extracellular matrix proteins5. The application of orthodontic forces can activate TGF-β1 transcription in these regions, thereby increasing TGF-β1 levels in GCF17. TGF-β1 can recruit osteoblasts precursors in the area of bone formation and stimulate osteoblasts to differentiate to produce bone matrix. During tooth movement, latent TGF-β1 is activated in the periodontal tissue by plasmin, which is produced by retracted periodontal ligament cells. During bone formation, TGF-β1 inhibits the recruitment of osteoclast precursor cells and directly inhibits osteoclast activity in bone resorption. Moreover, TGF-β1 in this area will stimulate osteoblastogenesis and new bone formation together with inhibition of osteoclastogenesis in this area5,16. Some researchers show that TGF-β1 can stimulate osteoclast apoptosis in the area. The effect of TGF β proapoptosis is mediated by several factors such as osteoblasts and stromal cells under TGF-β1 control5.\n\nThe phenolic content in virgin olive oil extracts such as apigenin, luteolin, coumaric acid, ferulic acid and affeic acid can increase the proliferation capacity and differentiation of osteoblasts. Inhibition of osteoclastogenesis is also seen in bone resorption markers, such as OPG(osteoprotegerin) and Receptor Activator of Nuclear Factor-KappaB Ligand (RANKL). The phenolic content in virgin olive oil extract can improve bone repair, and this is related to osteoblastogenesis and related factors, such as transcription factor 2 (RUNX-2), osterix (OSX), collagentype I (COL I) osteocalcin (OSC), and alkalinephosphatase (ALP). The phenolic compounds contained in virgin olive oil extract significantly increase the levels of TGFβ-1 and its receptors, and TGFβ-1 receptor expression is significantly increased in the presence of phenolic compounds17.\n\nTooth movement in orthodontic treatment can be divided into three stages: initial phase, lag phase, and post lag phase. In the initial phase, the orthodontic force will produce 0.4-0.9 mm of tooth movement. This phase takes place within one week. Cellular reaction starts after the application of force, this is seen by the presence of osteoclasts, osteoblasts, osteoblast progenitors and inflammatory cells. In the present study, TGFβ-1 levels of in the young and old guinea pigs reached the maximum value on observation day 7 indicating that the movement of the teeth is in the initial phase. Decreased levels of TGFβ-1 on day 14 indicate that tooth movement is in the lag phase. The lag phase is characterized by no or minimal movement of the teeth18.\n\n\nConclusion\n\nOlive oil increases TGFβ-1 levels in both young and old guinea pigs with tooth movement. Further studies are necessary at clinical levels to confirm the efficacy and potency of olive oil in increasing bone remodeling in orthodontic patients, especially in older patients.\n\n\nData availability\n\nFigshare: Picture1.jpg, https://doi.org/10.6084/m9.figshare.1009510119\n\nThis project contains the following underlying data:\n\nRaw data.xlsx (raw data of TGF beta levels among two groups tested)\n\nStatistic TGF beta.docs (statistic data)\n\nFig 1 (raw image used for Figure 1)\n\nFig 2 (raw image used for Figure 2)\n\nFig 3 (raw image used for Figure 3)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nDavidovitch Z, Nicolay OF, Ngan PW, et al.: Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am. 1998; 32(3): 411–35. PubMed Abstract\n\nKeeling SD, King GJ, McCoy EA, et al.: Serum and alveolar bone phosphatase changes reflect bone turnover during orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1993; 103(4): 320–6. PubMed Abstract | Publisher Full Text\n\nSeeman E: Bone modeling and remodeling. Crit Rev Eukaryot Gene Expr. 2009; 19(3): 219–33. PubMed Abstract | Publisher Full Text\n\nUdagawa N, Takahashi N, Jimi E, et al.: Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-kappa B ligand. Bone. 1995; 25(5): 517–23. PubMed Abstract | Publisher Full Text\n\nKobayashi Y, Hashimoto F, Miyamoto H, et al.: Force-induced osteoclast apoptosis in vivo is accompanied by elevation in transforming growth factor beta and osteoprotegerin expression. J Bone Miner Res. 2000; 15(10): 1924–34. PubMed Abstract | Publisher Full Text\n\nEhrman JK, Gordan PM, Visich PS, et al.: Clinical Exercise Physiolog. Human Kinetics, Inc, USA, 2013; 448. Reference Source\n\nRody WJ Jr, Wijegunasinghe M, Wiltshire WA, et al.: Differences in the gingival crevicular fluid composition between adults and adolescents undergoing orthodontic treatment. Angle Orthod. 2014; 84(1): 120–6. PubMed Abstract | Publisher Full Text\n\nKartiko BH, Siswanto FM: Hormon on anti aging medicine concept. J Virgin. 2015; 1(2): 108–22. Reference Source\n\nBerr C, Portet F, Carriere I, et al.: Olive oil and cognition: results from the three-city study. Dementia Geriatr Cogn Disord. 2009; 28(4): 357–364. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu H, Huang H, Li B, et al.: Olive oil in the prevention and treatment of osteoporosis after artificial menopause. Clin Interv Aging. 2014; 9: 2087–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuparwitri S, Pudyani PS, Haryana SM, et al.: Effects of soy isoflavone genistein on orthodontic tooth movement in guinea pigs. Dent J (Maj Ked Gigi). 2016; 49(3): 168–174. Publisher Full Text\n\nSuhardjono D: Percobaan hewan laboratorium. Gajah Mada University Press Yogyakarta, 1995; 207.\n\nTripoli E, Tabacchi G, Giammanco M, et al.: The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health. Nutr Res Rev. 2005; 18(1): 98–112. PubMed Abstract | Publisher Full Text\n\nKeiler AM, Zierau O, Bernhardt R, et al.: Impact of a functionalized olive oil extract on the uterus and the bone in a model of postmenopausal osteoporosis. Eur J Nutr. 2014; 53(4): 1073–81. PubMed Abstract | Publisher Full Text\n\nTuck KL, Hayball PJ: Major phenolic compounds in olive oil: metabolism and health effects. J Nutr Biochem. 2002; 13(11): 636–44. PubMed Abstract | Publisher Full Text\n\nChen G, Deng C, Lil YP: TGF-β1 and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci. 2012; 8(2): 272–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMelguizo-Rodríguez L, Manzano-Moreno FJ, Illescas-Montes R, et al.: Bone Protective Effect of Extra-Virgin Olive Oil Phenolic Compounds by Modulating Osteoblast Gene Expression. Nutrients. 2019; 11(8): pii: E1722. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlhasyimi AA, Pudyani PS, Asmara W, et al.: Effect of carbonated hydroxyapatite incorporated advanced platelet rich fibrin intrasulcular injection on the alkaline phosphatase level during orthodontic relapse. AIP Conf Proc. 2018; 1933: 030006. Publisher Full Text\n\nSuparwitri S: Picture1.jpg. figshare. Dataset, 2019. http://www.doi.org/10.6084/m9.figshare.10095101"
}
|
[
{
"id": "57888",
"date": "13 Jan 2020",
"name": "Ervina Sofyanti",
"expertise": [
"Reviewer Expertise Dentistry especially related to orthodontics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis title of manuscript has described the main question that addressed by this study. It is relevant and interesting topic in related to relapse of orthodontic treatment. It is necessary to have a scientific background that can show the common thread between previous researches and requirement of adequate orthodontic treatment (including relevant references for previous work and time citation) to understand the motivation and context of research by explaining the experimental approaches and reasons. Such as: The reason of using olive oil as sample is not too clear to prove why the authors using olive oil as sample to increase TGF-B expression in crevicular fluid, and the amount of crevicular fluid that is taken should be explained more detail.\n\nEven though the text is clear and easy to be read, this manuscript may require more adequate well-written English and alert to citations. Be careful in citation writing and bibliography. Then, I suggest to re-consider few references in introduction and discussion. Then explaining any steps taken to minimise the effects of subjective bias when allocating animals to treatment and describe who was blinded and when assessing results in this study.\n“Some researchers show that TGF-β can stimulate osteoclast apoptosis in the area. The effect of TGF-β pro apoptosis is mediated by several factors such as osteoblasts and stromal cells under TGF-β1 control5.” I recommend adding other references. Then I also recommend to re-consider the year of references and add some references in discussion.\n\nFinally, we recommend to revise this manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly",
"responses": [
{
"c_id": "5166",
"date": "07 May 2020",
"name": "Sri Suparwitri",
"role": "Author Response",
"response": "Dear Dr. Ervina and Dr. Denny Satria Thank you very much for your kind assistance in reviewing our manuscript. According to your minor suggestions, we have revised within the material and method section. This title of the manuscript has described the main question addressed by this study. It is a relevant and interesting topic related to the relapse of orthodontic treatment. It is necessary to have a scientific background that can show the common thread between previous researches and the requirement of adequate orthodontic treatment (including relevant references for previous work and time citation) to understand the motivation and context of research by explaining the experimental approaches and reasons. Such as The reason for using olive oil as the sample is not too clear to prove why the authors using olive oil as a sample to increase TGF-B expression in crevicular fluid, and the amount of crevicular fluid that is taken should be explained more detail. Thank you for your kind suggestion. We already added some information about it in the present manuscript. “Food ingredients that contain antioxidants can inhibit the oxidation process in the aging process. The oxidation process can increase intracellular reactive oxygen activity so that it can inhibit bone remodeling. Olive oil is a food ingredient that contains a lot of antioxidants and is often consumed by the public7. Olive oil is extracted from fresh olives have been shown to slow the aging process8. Accord ing to Liu et al., olive oil acts effectively as a substitute for estrogen to prevent bone loss9. According to Rodrigues et al., The phenolic compound in virgin olive oil extracts can increase the proliferation and differentiation of osteoblasts. Phenolic compounds (caffeic acid, ferulic acid, coumaric acid, apigenin, and luteolin) in olive oil extracts can increase TGF-β1 and its receptor7.” Even though the text is clear and easy to be read, this manuscript may require more adequate well-written English and alert to citations. Be careful in citation writing and bibliography. Then, I suggest reconsidering a few references in the introduction and discussion. Then explaining any steps taken to minimise the effects of subjective bias when allocating animals to treatment and describe who was blinded and when assessing results in this study. We already added some information about it: “Sample groups were selected using simple random number sampling. Each animal was assigned a unique number, which was picked out of a box by a blindfolded researcher.” “Some researchers show that TGF-β can stimulate osteoclast apoptosis in the area. The effect of TGF-β pro apoptosis is mediated by several factors such as osteoblasts and stromal cells under TGF-β1 control5.” I recommend adding other references. Then I also recommend to re-consider the year of references and add some references in the discussion. We already added some information about it: “Previous study show that TGF-β1 can stimulate osteoclast apoptosis in the area. The effect of TGF β proapoptosis is mediated by several factors such as osteoblasts and stromal cells under TGF β1 control 3.We also have added and revised our references in the present manuscriptThank you for the reviewRegards"
},
{
"c_id": "5475",
"date": "07 May 2020",
"name": "Sri Suparwitri",
"role": "Author Response",
"response": "Dear Dr Mingyuan Du and Dr Hong He Thank you for your kind assistance in reviewing our manuscript and providing us with valuable advice for the manuscript and our future work. We have been working throughout the review. The following are our consolidated comments related to the review and some changes we have done based on the reviewer comments. At first, several references of publications should be added in Discussion. For example, “TGF-β1 can recruit osteoblasts precursors in the area of bone formation and stimulate osteoblasts to differentiate to produce bone matrix” and “During bone formation, TGF-β1 inhibits the recruitment of osteoclast precursor cells and directly inhibitsosteoclast activity in bone resorption.” And please check full of the manuscript. We have edited the discussion section and added other references, Thank you for the punctiliously review. We have added this information in the present manuscript In this paper, the number of guinea pigs used in the current experiments is 12. I want to know how did you calculate the required number size and why? We count it based on the Lemeshow's formula (t-1)(n-1)≥ 15 (7-1)(n-1) ≥15 n ≥ 3,5 t represent a total group we determined on this study and n represent the sample. From those formula we have got 3.5 and base on animal restriction we decided to round numbered down into three samples for each group In addition, as the animals in Olive oil treatment groups were treated with olive oil, were the animals in the control group treated with mock oil? Thank you for the punctiliously review. We have added this information in the present manuscript Finally, please add more details about methods including Isolation of the GCF and Orthodontic installation in order for peers to reproduce your research. We have edited the methods section and added this information in the present manuscript. We thank you again for the valuable insight."
}
]
},
{
"id": "60725",
"date": "09 Mar 2020",
"name": "Mingyuan Du",
"expertise": [
"Reviewer Expertise Mechanisms of orthodontic tooth movement"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper titled \"Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs \" showed that TGF-β1 levels in the younger age guinea pigs were higher than the older age, and olive oil could increase TGF-β1 levels in the older age guinea pigs.\n\nHere are a few questions:\nAt first, several references of publications should be added in Discussion. For example, “TGF-β1 can recruit osteoblasts precursors in the area of bone formation and stimulate osteoblasts to differentiate to produce bone matrix” and “During bone formation, TGF-β1 inhibits the recruitment of osteoclast precursor cells and directly inhibitsosteoclast activity in bone resorption.” And please check full of the manuscript.\n\nIn this paper, the number of guinea pigs used in the current experiments is 12. I want to know how did you calculate the required number size and why?\n\nIn addition, as the animals in Olive oil treatment groups were treated with olive oil, were the animals in the control group treated with mock oil?\n\nFinally, please add more details about methods including Isolation of the GCF and Orthodontic installation in order for peers to reproduce your research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5475",
"date": "07 May 2020",
"name": "Sri Suparwitri",
"role": "Author Response",
"response": "Dear Dr Mingyuan Du and Dr Hong He Thank you for your kind assistance in reviewing our manuscript and providing us with valuable advice for the manuscript and our future work. We have been working throughout the review. The following are our consolidated comments related to the review and some changes we have done based on the reviewer comments. At first, several references of publications should be added in Discussion. For example, “TGF-β1 can recruit osteoblasts precursors in the area of bone formation and stimulate osteoblasts to differentiate to produce bone matrix” and “During bone formation, TGF-β1 inhibits the recruitment of osteoclast precursor cells and directly inhibitsosteoclast activity in bone resorption.” And please check full of the manuscript. We have edited the discussion section and added other references, Thank you for the punctiliously review. We have added this information in the present manuscript In this paper, the number of guinea pigs used in the current experiments is 12. I want to know how did you calculate the required number size and why? We count it based on the Lemeshow's formula (t-1)(n-1)≥ 15 (7-1)(n-1) ≥15 n ≥ 3,5 t represent a total group we determined on this study and n represent the sample. From those formula we have got 3.5 and base on animal restriction we decided to round numbered down into three samples for each group In addition, as the animals in Olive oil treatment groups were treated with olive oil, were the animals in the control group treated with mock oil? Thank you for the punctiliously review. We have added this information in the present manuscript Finally, please add more details about methods including Isolation of the GCF and Orthodontic installation in order for peers to reproduce your research. We have edited the methods section and added this information in the present manuscript. We thank you again for the valuable insight."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2028
|
https://f1000research.com/articles/9-337/v1
|
07 May 20
|
{
"type": "Research Article",
"title": "Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometry",
"authors": [
"Milena Music",
"Marco Iafolla",
"Antoninus Soosaipillai",
"Ihor Batruch",
"Ioannis Prassas",
"Melania Pintilie",
"Aaron R. Hansen",
"Philippe L. Bedard",
"Stephanie Lheureux",
"Anna Spreafico",
"Albiruni Abdul Razak",
"Lillian L. Siu",
"Eleftherios P. Diamandis",
"Milena Music",
"Marco Iafolla",
"Antoninus Soosaipillai",
"Ihor Batruch",
"Ioannis Prassas",
"Melania Pintilie",
"Aaron R. Hansen",
"Philippe L. Bedard",
"Stephanie Lheureux",
"Anna Spreafico",
"Albiruni Abdul Razak",
"Lillian L. Siu"
],
"abstract": "Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB. Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs. Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8–173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1–32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2–17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0–3.2, p=0.05). Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.",
"keywords": [
"programmed cell death protein 1",
"immune checkpoint blockade",
"pembrolizumab",
"response",
"predictive biomarkers",
"autoantibodies",
"anti-thyroglobulin antibody",
"anti-thyroid peroxidase antibody",
"hypothyroidism",
"immune-related adverse events"
],
"content": "List of abbreviations\n\nABC, ammonium bicarbonate; CBR, clinical benefit rate; CI, confidence intervals; CR, complete response; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; FDR, false discovery rate; HR, hazard ratios; ICB, immune checkpoint blockade; irAE, immune related adverse event; MPS, modified percent score; NSCLC, non-small cell lung cancer; OR, odds ratios; OS, overall survival; PD-1, programmed cell death protein 1; PFS, progression-free survival; PR, partial response; SD, stable disease; Tg, thyroglobulin; TPO, thyroid peroxidase.\n\n\nIntroduction\n\nDespite the ability of immune checkpoint blockade (ICB) to produce enduring responses in several advanced malignancies1,2, there are several limitations. Monotherapy response rates for inhibitors of programmed cell death protein-1 or its ligand (anti-PD-1/L1) across solid tumors are generally modest at approximately 20–30%, and much lower in some tumor types3,4. All-grade and high-grade toxicities with PD-1/L1 approach 40% and 12%, respectively5–8. Hence, there is an unmet need for predictive biomarkers to enable patient selection. Although numerous markers have already been tested for their ability to predict response to ICB1, many require molecular and immune profiling from either invasive biopsy or archival tumor specimen9. Similarly, validated biomarkers that can identify patients who develop immune-related adverse events (irAEs) to ICB are lacking.\n\nAntigen spreading is the phenomenon of in situ damaged cancer cells releasing intracellular antigens into the surrounding environment10,11. Although tumorigenesis distorts the native structure of these antigens12, which in turn elicit a humoral immune response that produces tumor associated antigen autoantibodies13–16, several studies have investigated the biomarker potential of autoantibodies targeting wild-type proteins using large human proteome arrays in patients treated with ICB. For instance, castrate resistant prostate cancer patients responding to ipilimumab demonstrate an increase in autoantibodies targeting intracellular proteins (such as MPG, PAK6 and DLX1) relative to non-responders17. Melanoma patients who developed severe irAEs from the combination of intralesional bacillus Calmette-Guérin injection and ipilimumab were found to have a significant increase in autoantibodies directed against self-antigens that preceded the clinical diagnosis of toxicity18. Pre-treatment autoantibody profiles in melanoma patients treated with anti-PD-1, anti-CTLA-4 or a combination of both agents were unique to those who developed severe irAEs19. Humoral immunity may be increased via PD-1 inhibition through direct interaction with PD-1 receptors on B-cells20 and/or non-cytotoxic T-cell dependent processes21.\n\nCurrent attempts to characterize serum autoantibodies are limited by analytic constraints inherent to complex biological samples, such as blood or lysate, that are fraught with low sensitivity22,23. This study attempts to overcome this analytic limitation by typifying the serum autoantibodies using a modified version of the established Serologic Proteome Analysis assay24–26 that incorporates protein G purification of serum autoantibodies and tandem mass spectrometric identification of the antibody targets.\n\nWe hypothesize that the magnitude of autoantibody production is a measure of response and/or toxicity to ICB. Our primary objective is to isolate candidate serum autoantibodies from advanced solid tumor patients receiving pembrolizumab (anti-PD-1) monotherapy on a prospective phase II clinical trial and correlate their titers with clinical outcomes.\n\n\nMethods\n\nThe single-centre investigator-initiated biomarker phase II clinical trial called INSPIRE (INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation; ClinicalTrials.gov ID NCT02644369, registered on 31 December 2015) was designed to analyze dynamic changes in genomic, proteomic and immunologic landscapes in patients treated with pembrolizumab (anti-PD-1 antibody) monotherapy. Between March 21, 2016 to May 9, 2018, patients were prospectively enrolled into one of the following five cohorts: squamous cell cancer of the head and neck, triple negative breast cancer, epithelial ovarian cancer, malignant melanoma (cutaneous and non-cutaneous) or mixed solid tumors. Approximately 20 patients were enrolled into each cohort; the INSPIRE study was purely exploratory and no formal sample size was required. Pertinent inclusion criteria: age ≥18 years; ECOG 0-1; incurable histologically proven locally advanced or metastatic solid malignancy without further standard treatment options (with the exception of melanoma); measurable disease based on RECIST 1.127; and adequate organ functions. Pertinent exclusion criteria: prior anti-PD-1/L1/L2 agents (prior anti-CLTA4 and T-cell co-stimulatory agents allowed); autoimmune disease; immunodeficiency or immunosuppressive medications exceeding physiologic corticosteroid replacement; leptomeningeal disease; or <4 weeks of stable central nervous system metastases.\n\nAll patients received monotherapy pembrolizumab (anti-PD-1 antibody) 200 mg IV every 3 weeks, were clinically assessed with comprehensive blood work every 3 weeks and received restaging CT scans every 9 weeks. Clinical benefit rate (CBR) was defined a priori as RECIST 1.1 complete response (CR) or partial response (PR), or stable disease (SD) for ≥6 cycles of pembrolizumab. Toxicity was defined a priori as ≥Grade 2 CTCAE 4.0328 irAE with at least possible attribution to pembrolizumab of which the investigator asserted the adverse event to be autoimmune in causality from pembrolizumab exposure. Grade 2 or higher grade irAEs are considered clinically significant29. Only the highest grade of a specific toxicity event was recorded for each patient; the same toxicity event was recorded more than once per patient if the irAE re-occurred on a separate occasion (i.e. not a re-flare during immunosuppression taper). Both response and toxicity data were annotated by INSPIRE data coordinators and then verified by one author (MI). The causality of irAE to pembrolizumab was reviewed by MI and LLS.\n\nThe Research Ethics Board located at Princess Margaret Cancer Centre, University Health Network (Toronto, Canada) reviewed and approved this study (#15-9828). All patients provided written informed patient consent prior to study enrollment. The study was carried out in accordance with the Declaration of Helsinki.\n\nBlood samples for this analysis were drawn within 28 days prior to first pembrolizumab exposure (baseline) and 7 weeks into treatment with pembrolizumab (pre-cycle 3). Upon collection of blood samples in Vacutainer® SST™ tubes, the blood was allowed to clot for 30–60 minutes, and the tubes were then centrifuged at 1,200g for 10 minutes at room temperature. In total, 500 μL of serum was aliquoted into cryogenic vials and frozen at -80°C.\n\nIn order to optimize autoantibody identification, 24 patients treated on INSPIRE were selected by MI and LLS and placed into one of four groups: 6 patients without CBR or toxicity from pembrolizumab (Group I); 6 patients with toxicity but no CBR (Group II); 6 patients with both CBR and toxicity (Group III); and 6 patients with CBR but no toxicity (Group IV). To minimize bias, performance of the experiments and analysis of the results were conducted by investigators blinded to INSPIRE clinical outcomes.\n\nIn total, 12 µL of serum from each of the 6 individual patients in the four Groups (I–IV) were pooled to create mixed pools (72 µL/pool). This was done for the serum samples collected both before and after immunotherapy initiation, resulting in the following eight pooled serum samples: Group I (baseline), Group I (pre-cycle 3), Group II (baseline), Group II (pre-cycle 3), Group III (baseline), Group III (pre-cycle 3), Group IV (baseline) and Group IV (pre-cycle 3).\n\nHuman tissues were obtained from Mount Sinai Hospital (REB# 18-0077-E) and the University Health Network (REB #15-9680) at autopsy or during surgical removal. Tissue samples were stored at -80° C until ready for use. Protein was extracted from a total of 29 human body tissues and 4 human brain-specific tissues for comprehensive proteome coverage (see Extended data30). For protein extraction, tissue was pulverized in liquid nitrogen with a mortar and pestle to yield a fine powder. Next, 0.2% RapiGest SF Surfacant (Waters, Milford, MA, USA) in 50 mM ammonium bicarbonate (ABC) was added to further lyse the tissues. The pulverized tissue sample was vortexed every 5–10 min on ice, for 30 min, and then sonicated on ice for 15 sec, three times, to further disrupt the cells. Following sonication, the sample was centrifuged at 15,000g for 20 min at 4°C, to remove debris and insoluble contents, followed by collection of the supernatant. A Pierce BCA Protein Assay (Thermo Fisher Scientific, San Jose, California) was performed on tissue lysate protein extracts for total protein quantification. Equal amounts of each tissue lysate were combined to make a 1.5-mg complex tissue lysate for each immunoprecipitation experiment.\n\nDetails are stated in Extended data30. A similar method was recently validated in a proof-of-concept study that included autoantibodies to CUB and zona pellucida-like domain-containing protein 1 and pancreatic secretory granule membrane major glycoprotein 2 in the sera of patients with inflammatory bowel disease31.\n\nThe autoantibody protein targets identified before and after pembrolizumab treatment in Groups I through IV were compared. Immunoglobulins, keratins, serum albumin and other non-specific serum-abundant proteins (such as hemoglobin), as well as complement proteins and apolipoproteins that are normally immunoprecipitated as part of immune complexes in the blood32, were excluded. The candidates were chosen based on the a priori definition of ≥2-fold peptide number increase for the target protein from pre- to post-treatment serum in patient group II, III or IV (n=6 in each group) and either a ≥4-fold peptide number difference compared to patient group I (n=6) or no identified peptides in group I17. Due to cost and feasibility, only the top candidate and a related protein candidate were further evaluated by electro-chemiluminescence immunoassay.\n\nImmuno-mass spectrometric analysis of the discovery set showed that thyroglobulin (Tg) was the top candidate autoantibody target that met the pre-defined selection criteria. Thyroid peroxidase (TPO) was chosen as an additional candidate autoantibody target due to its known association with anti-Tg antibody in autoimmune disease. Immuno-mass spectrometric results are available as Underlying data33.\n\nAll INSPIRE patients with baseline and pre-cycle 3 sera (including those used in the discovery analysis) were analyzed for the candidate autoantibody titers by electrochemiluminescence immunoassays (Elescys Anti-Tg and Elecsys Anti-TPO, Roche Diagnostics, Risch-Rotkreuz, Switzerland) using a Cobas e 411 analyzer. The Cobas e 411 analyzer has an approximate 10% coefficient of variation for both anti-Tg34 and anti-TPO35 titers. Further, the coefficient of biological variation of these antibodies is also approximately 10%36. To be conservative, a minimum 50% increase in titers from baseline to pre-cycle 3 was used to define a significant increase. Based upon instrument limitations, the measurable range of anti-TPO and anti-Tg antibodies were 5.00–600 IU/mL and 10.0–4000 IU/mL, respectively. Patients with anti-TPO titers <5.00 IU/mL and anti-Tg titers < 10.0 IU/mL were arbitrarily assigned 5.00 IU/mL and 10.0 IU/mL, respectively. One patient in the mixed solid tumor cohort had an anti-Tg titer >4000 IU/mL both at baseline and cycle 3; the value of 4000 UI/mL was used for both time points. The median value of anti-Tg titers at both baseline and pre-cycle 3 was 10.0 IU/mL (range: 10.0–4000 IU/mL) and the median value of anti-TPO titers at baseline and pre-cycle 3 was 10.10 IU/mL and 10.70 IU/mL, respectively (range: 5.00–434.70 IU/mL). For simplicity, we defined elevated anti-Tg and anti-TPO when the titer was > 10.0 IU/mL. The investigators conducting all experiments and analysis of the results were blinded to INSPIRE clinical outcomes.\n\nThe co-primary endpoints were CBR and toxicity as defined above. Patients were dichotomized at baseline and pre-cycle 3 using median titers of candidate autoantibodies, and the change (Δ) in autoantibody titer was defined as a 1.5x increase in titer from baseline to pre-cycle 3 versus stable or decrease in titer (only patients who had pre-cycle 3 titers >10.0 IU/mL were included in the Δ analysis). These groups were analyzed as predictors of CBR and toxicity. Due to the possible influence of toxicity on autoantibody production, patients who developed toxicity before the pre-cycle 3 blood draw were removed from both the CBR and toxicity pre-cycle 3 and Δ analysis. Statistical significance among the categorical variables was evaluated using Fisher’s exact test. Further, the individual patient titers at baseline, pre-cycle 3, and difference in titer (defined as pre-cycle 3 titer minus baseline titer) were explored as continuous variables using the Mann-Whitney test. As with the categorical variable analysis above, patients who developed toxicity before the pre-cycle 3 blood draw were removed from both the CBR and toxicity pre-cycle 3 and difference in titer analysis.\n\nIn an effort to reduce confounding in our observational study with a small number of events, propensity scores were created based on a model incorporating age, gender, ethnicity and PD-L1 status. As outlined in the interim INSPIRE trial report37, this study used formalin-fixed paraffin-embedded blocks from screening biopsies. The PD-L1 immunohistochemistry clone 22C3 was applied to 4–5 μm sections mounted on positively charged ProbeOn slides (QualTek, Goleta, CA). QualTek produced a modified proportion score (MPS) denoting the proportion of PD-L1-expressing tumor cells and mononuclear inflammatory cells within tumor nests. Further details are found in Extended data30. Conditional logistic regression was applied to test each candidate autoantibody and their change adjusting for strata and the propensity scores. Odds ratios (OR), their 95% confidence intervals (CI) and p-values were thus obtained.\n\nOverall survival (OS) and progression free survival (PFS) times were calculated as the durations between the first infusion with pembrolizumab and death or progression, respectively. When death or progression were not observed the patient was considered censored. The survival percentages and median survival estimates were calculated using Kaplan-Meier method. The Hazard Ratios (HR) and their CIs were calculated within the Cox Proportional-Hazards Model. The association of toxicity with OS and PFS was analyzed using toxicity as a time-dependent covariate. The association between covariates and candidate autoantibodies were investigated using Fisher’s exact test for categorical variables, or Mann-Whitney test or Spearman correlation for continuous variables (where appropriate).\n\nAll p-values were two-sided and p-values < 0.05 were considered statistically significant.\n\nAll calculations were performed using R 3.4 (The R Foundation for Statistical Computing).\n\n\nResults\n\nIn total, 106 patients were enrolled into INSPIRE and 78 patients were included in this analysis. Among the excluded 28 INSPIRE patients: 2 patients had both baseline and pre-cycle 3 serum blood work but the pre-cycle 3 serum was not processed for autoantibody quantification; 17 patients were taken off trial between cycle 2 and cycle 3; and 9 patients were taken off trial before cycle 2. Deidentified patient outcomes are available as Underlying data33.\n\nThe 24 patient samples used in the discovery analysis were frozen for a median of 566 days (range 335–835 days) and thawed twice. All 78 patients used in the test set (which included the 24 patients in the discovery analysis) were frozen for a median of 626.5 days (range 257–927 days) and thawed once.\n\nTable 1 summarizes pertinent clinicopathologic information and their association with CBR and toxicity. Gender was approximately evenly distributed (female 55%, male 45%). The median age was 61 years at time of first pembrolizumab infusion, but there was a large range in age (21–82 years). The vast majority of patients were of white ethnicity (n=64, 83%). Head and neck squamous cell carcinoma (n=14, 18%) comprised the major tumor type. Median PD-L1 MPS were 1% (range: 0 – 100%). Unadjusted univariate analysis suggested the response rate was different between the cohorts (p=0.011) and the patients who had CBR tended to have a higher percent of PD-L1 MPS in comparison to those without CBR (medians 1.5% vs 0%, p=0.029).\n\nOf the entire INSPIRE cohort (n=106), 28 patients did not have paired baseline and pre-cycle 3 serum for analysis and thus were excluded.\n\nCBR = clinical benefit rate; HGSOC = high-grade serous ovarian carcinoma; HNSCC = head and neck squamous cell carcinoma; MPS = modified percent score; TNBC = triple negative breast cancer.\n\na P-value calculated by Fisher Exact Test, unadjusted.\n\nb P-value calculated by Mann-Whitney, unadjusted.\n\nThe CONSORT diagram in Figure 1 depicts the relevant patient and serum sample flow through this study and clinical outcomes of each cohort. Last clinical outcome update was May 3, 2019; at that time the median follow-up was 2.35 years from date of first pembrolizumab infusion (range: 0.79–2.95 years), two patients (2.6%) were lost to follow up and 49 deaths (63%) occurred. Of the two patients lost to follow up, both developed disease progression as best response and one developed a significant toxicity event; both patients were included in the analysis. The median number of pembrolizumab infusions before coming off trial was five (range: 2–35 infusions). All patients were evaluable. CBR was achieved in 24 patients (31%): CR, PR and SD ≥6 cycles of pembrolizumab occurred in 3 patients (3.9%), 13 patients (17%) and 8 patients (10%), respectively. In total, three patients (3.9%) remained on treatment at time of analysis. RECIST disease progression (n=48, 62%) was the major reason for stopping treatment; five patients (6.4%) completed all 35 pembrolizumab cycles specified in the trial. Toxicity occurred in 19 patients (24%); hypothyroidism (n=8, 10%) was the most common toxicity, and nine patients (12%) developed more than one toxicity. Median number of days from first dose of pembrolizumab to toxicity onset was 105 (range: 1–482) and five patients (6.4%) developed toxicity before pre-cycle 3 blood draw. Toxicity necessitated stopping treatment in five patients (6.4%); of these, Grade 3 colitis (n=2, 2.6%) and Grade 3 pneumonitis (n=2, 2.6%) were the most common events. In total, seven patients (9.0%) developed Grade 3 toxicity, two patients (2.6%) developed more than one Grade 3 event and all remaining toxicity events were Grade 2; there were no Grade 4 or 5 events.\n\nNote that patients can develop more than one type of toxicity event. HNSCC, head and neck squamous cell carcinoma; TNBC, triple-negative breast cancer; HGSOC, high-grade serous ovarian carcinoma; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; CBR, clinical benefit rate; ALT, alanine aminotransferase; AST, aspartate aminotransferase.\n\nOf the 24 patients used in the discovery pooled serum analysis, melanoma (n=9; 38%) and ovarian cancer (n=5; 21%) comprised the majority. Two patients who were initially assigned to Group IV (+CBR / -toxicity) later developed immune-related hypothyroidism. There were 14 patients with toxicity in this discovery analysis; the most common events were hypothyroidism (n=6, 43%), rash (n=3, 21%) and pneumonitis (n=3, 21%). In total, four patients developed toxicity before the pre-cycle 3 blood draw. The protein intensities of Tg and TPO identified by immuno-mass spectrometry in the four patient group discovery analysis at both baseline and pre-cycle 3 are shown in Figure 2a and Figure 2b, respectively.\n\nThe average baseline and pre-cycle 3 thyroglobulin (Tg) (a) and thyroid peroxidase (TPO) (b) protein intensities. We identified Tg and TPO as candidates during autoantibody target selection from pooled patient sera. CBR, clinical benefit rate.\n\nOf all 78 patients included in this analysis, at baseline, 20 patients had both elevated anti-Tg and anti-TPO titers, three patients had only elevated anti-Tg titers, and 19 had only elevated anti-TPO titers. At pre-cycle 3, 24 patients had both elevated anti-Tg and anti-TPO titers, two patients had only elevated anti-Tg titers, and 18 had only elevated anti-TPO titers. In total, 10 and eight patients developed a significant Δ in anti-Tg and anti-TPO titers, respectively. No patients developed ≥50% decrease in titer from baseline to pre-cycle 3. De-identified patient autoantibody titres are available as Underlying data33.\n\nAnti-Tg (Table 2) and anti-TPO (Table 3) titers at baseline, pre-cycle 3 and Δ were analyzed for their association with CBR and toxicity. The analysis of the dichotomized values suggested some associations between anti-Tg and toxicity: Δ was significant in unadjusted (p=0.0024), cohort adjusted (OR=23.8, 95% CI 2.6–221.5, p=0.0053), and multivariable models (OR=17.4, 95% CI 1.8–173.8, p=0.015), suggesting an increased risk for toxicity for patients with a pre- anti-Tg cycle 3 titer ≥ 1.5x from baseline. Higher levels of anti-Tg pre-cycle 3 also predicted for higher rate of toxicity when adjusting for cohort (OR=4.7, 95% CI 1.2–17.8, p=0.024). Similarly, anti-TPO Δ was significant for predicting toxicity in patients with ≥1.5x increase in anti-TPO titers from baseline to pre-cycle 3 in unadjusted (p=0.039) and cohort adjusted (OR=6.1, 95% CI 1.1–32.7, p=0.035) models, although significance was lost in multivariable analysis (p=0.078). Anti-Tg titers at baseline and pre-cycle 3 were not significant for predicting toxicity in multivariate analysis, and anti-TPO baseline and pre-cycle 3 titers were not significant for predicting toxicity in any of the analyses. Further, anti-Tg and anti-TPO titers were not associated with CBR at any time point regardless of statistical approach.\n\nBaseline and pre-cycle 3 categories were dichotomized by median titers (10.0 IU/mL) versus > median titers (10.0 IU/mL was anti-Tg titer's lowest limit of instrument detection). Δ was dichotomized using pre-cycle 3 titers ≥ 1.5x baseline titers vs pre-cycle 3 titers < 1.5x baseline titers. Patients who developed toxicity before pre-cycle 3 blood draw (n=5) were removed from both the CBR and toxicity pre-cycle 3 and Δ analysis.\n\nCBR = clinical benefit rate; CI = confidence intervals; OR = odds ratio.\n\naDetermined using propensity scores with cohort as strata and the following covariates: age, gender, ethnicity and PD-L1 status. One patient was removed due to unknown ethnicity.\n\nbCalculated using Fisher’s Exact Test.\n\ncBased on conditional logistic regression with the cohort as strata.\n\ndBased on conditional logistic regression with the cohort as strata and adjusted for the propensity scores.\n\nBaseline and pre-cycle 3 categories were dichotomized by ≤ to median titers (10.0 IU/mL) versus > median titers. Δ was dichotomized using pre-cycle 3 titers ≥ 1.5x baseline titers vs pre-cycle 3 titers < 1.5x baseline titers. Patients who developed toxicity before pre-cycle 3 blood draw (n=5) were removed from both the CBR and toxicity pre-cycle 3 and Δ analysis.\n\nCBR = clinical benefit rate; CI = confidence intervals; OR = odds ratio.\n\naDetermined using propensity scores with cohort as strata and the following covariates: age, gender, ethnicity and PD-L1 status. One patient was removed due to unknown ethnicity.\n\nbCalculated using Fisher’s Exact Test.\n\ncBased on conditional logistic regression with the cohort as strata.\n\ndBased on conditional logistic regression with the cohort as strata and adjusted for the propensity scores.\n\nAnti-Tg and anti-TPO titers were also explored as continuous variables for their possible association with CBR and toxicity. The Mann-Whitney test (unadjusted analysis) showed an association of elevated anti-Tg titers with higher risk of toxicity at baseline (p=0.043), pre-cycle 3 (p=0.011) and the difference in titer between these two time points (p=0.001) (Extended data30); significance was lost in cohort adjusted and multivariable models. The same type of association was found with anti-TPO: elevated anti-TPO titers were associated with higher risk of toxicity at pre-cycle 3 in the unadjusted model (p=0.045) and difference in titers between baseline and pre-cycle 3 in the cohort adjusted model (p=0.05), although significance was lost in multivariable analysis (Extended data30). When analyzed as a continuous variable, anti-Tg and anti-TPO were not predictive of CBR (Extended data30).\n\nPrior to trial enrolment, three patients had pre-existing thyroid disease: two patients with hypothyroidism both had elevated anti-TPO titers at baseline and/or pre-cycle 3 and the one patient with thyroid nodules had non-elevated titers; none of these patients developed either CBR or toxicity on trial. Hypothyroidism development during trial (n=8) was more common among: patients with elevated anti-Tg titers at baseline (75% vs 24%, p=0.007), pre-cycle 3 (86% vs 27%, p=0.0044) and increase in titers between these two time points (71% vs 7%, p=0.00026); and in patients with an increase in anti-TPO titer between baseline and pre-cycle 3 (57% vs 7%, p=0.0029). Of the eight patients who developed hypothyroidism, six (75%) had both elevated anti-Tg and anti-TPO antibodies at baseline and pre-cycle 3, one patient (12.5%) developed elevated anti-Tg and anti-TPO titers at pre-cycle 3, and the last patient had non-elevated titers at both time points. Conversely, of the 70 patients who did not develop hypothyroidism, 42 (60%) had elevated anti-Tg and/or anti-TPO titers at baseline and/or pre-cycle 3. There was no statistically significant association of anti-Tg or anti-TPO with the development of non-thyroid toxicities. Of the 11 patients who developed non-thyroidal toxicity, five (46%) had either anti-Tg or anti-TPO titers elevated at baseline or pre-cycle 3.\n\nElevated baseline anti-Tg or anti-TPO titers did not show a significant difference in OS (Figure 3a and Figure 4a, respectively) or PFS (see Extended data30). Although elevated pre-cycle 3 anti-Tg titers were not associated with OS (Figure 3b), patients with elevated pre-cycle 3 anti-TPO titers did have a significant difference in OS (23.8 months vs 11.5 months; HR=1.8, 95% CI 1.0–3.2, p=0.05) (Figure 4b). Pre-cycle 3 anti-Tg and anti-TPO titers were not associated with PFS (see Extended data30). Finally, anti-Tg and anti-TPO Δ was not significant for OS and PFS (see Extended data30).\n\nPatients were dichotomized based upon anti-thyroglobulin (Tg) titers greater than, or equal to, median (10.0 IU/mL) at (a) baseline and (b) pre-cycle 3. B, baseline; C3, pre-cycle 3; CI, confidence interval; HR, hazard ratio.\n\nPatients were dichotomized based upon anti-TPO levels greater than median (10.0 IU/mL) or ≤ median at (a) baseline and (b) pre-cycle 3. P-values are unadjusted and calculated using the Wald test. B, baseline; C3, pre-cycle 3; CI, confidence interval; HR, hazard ratio.\n\nToxicity analysed as a time-dependent covariate was not associated with OS (p=0.11) or PFS (p=0.47).\n\nAssociations between the candidate autoantibodies were explored as both categorical and continuous variables with the confounding covariates: age, gender, ethnicity and PD-L1 status. White ethnicity was associated with higher anti-Tg titers at baseline (continuous: p=0.048) and pre-cycle 3 (categorical: p=0.05; continuous: p=0.031) (see Extended data30). The remaining covariates did not exhibit any association with the autoantibodies (see Extended data30). The Spearman correlation coefficients calculated between the autoantibodies with age and PD-L1 as continuous variables were very small ranging between -0.167 and 0.14, suggesting lack of association between these variables (see Extended data30).\n\n\nDiscussion\n\nTo our knowledge, this is the first study to perform an extensive autoantibody analysis in the pre- and post-pembrolizumab sera of patients with mixed solid tumors, and then determine the association of candidate autoantibodies with clinical benefit and toxicity. Our study suggested that patients with an increase in anti-Tg or anti-TPO titers from baseline to pre-cycle 3 are associated with significant toxicity in unadjusted and cohort-adjusted models, and the anti-Tg increase is also significant for toxicity in multivariable models. Hypothyroidism was observed in 10% in our cohort, which is consistent with the 6.5–7.9% incidence reported in an ICB meta-analysis38, and accounted for 42% of all our toxicity events. Nearly half of our patients with non-thyroid toxicity had elevated anti-Tg and/or anti-TPO titers; however, we were unable to demonstrate an association of these antibodies with non-thyroid toxicity. A recent retrospective analysis of non-small cell lung cancer (NSCLC) patients treated with anti-PD-1 monotherapy demonstrated similar results: pre-treatment anti-Tg and/or anti-TPO antibodies were associated with the development of immune-related hypothyroidism, but not other irAEs39. While antibodies for multiple autoimmune illnesses can develop simultaneously40–42, our analysis only chose the candidate with the largest number of identified peptides and a related candidate, and thus any non-thyroidal autoantibodies that may have been present were not analyzed. Future work assessing a larger spectrum of autoantibodies to create a biomarker signature will aid in addressing this issue.\n\nHerein we found an association between elevated pre-cycle 3 anti-TPO antibodies with OS. This is in keeping with the NSCLC study that showed baseline autoantibodies (including anti-Tg and anti-TPO) were associated with improved PFS and disease control rate during treatment with anti-PD-1 monotherapy39. However, given our lack of association between anti-TPO with CBR and PFS, and baseline and Δ anti-TPO with OS, caution must be used in interpreting this result. Future trials enriching for specific disease sites will help elucidate the role autoantibodies may play as a prognostic and predictive biomarker to ICB.\n\nThe association of irAEs with increased survival or response to ICB has been found in melanoma43 and NSCLC44 studies using statistical methods to prevent bias from the time-dependence of both predictor and outcome variables45,46. The analysis of toxicity as a time-dependent covariate did not corroborate this principle in our prospective trial of multiple histologies. If benefit is most likely procured from patients with toxicity events, then astute monitoring and early recognition of irAEs is paramount to control these toxicity events and prevent early treatment discontinuation.\n\nNot surprisingly, our association of baseline and on treatment anti-Tg or anti-TPO antibodies with the development of hypothyroidism is supported by other anti-PD-1 studies20,21,39,47. Interestingly, it appears cancer patients harbor higher rates of pre-existent autoantibodies: our pre-treatment combined anti-Tg and anti-TPO rate of 54%, and published rates of NSCLC patient pre-treatment rheumatoid factor and antinuclear antibody rates of 28% and 35%, respectively39, are higher than the general population rates of anti-Tg (11%), anti-TPO (13%), rheumatoid factor (5–25%) and antinuclear antibodies (27%)48–50. While the development of tumor-associated autoantibodies to aberrant protein structures12 has undergone considerable biomarker investigation51–55, the mechanism of cancer patients developing autoantibodies to endogenous proteins and their biomarker potential is less clear.\n\nThe increase in anti-thyroid antibody titers shortly after treatment initiation, or the development of new anti-thyroid antibodies following treatment, points to a possible unmasking of latent autoimmunity by pembrolizumab. Patients with pre-existing latent autoimmunity characterized by increased autoantibody titers may be at higher risk of developing toxicity from ICB. We suggest future prospective studies of other autoimmune markers at baseline are evaluated in patients treated with ICB to further elucidate a mechanism for this effect.\n\nOur study showed that patients can still develop hypothyroidism without the presence of anti-Tg and anti-TPO antibodies. Our reported rate on study of elevated anti-Tg and/or anti-TPO antibodies among those who developed hypothyroidism was 88%, which is comparable to the rates of elevated anti-Tg and anti-TPO antibodies in patients with chronic autoimmune thyroiditis within the general population (70–80% and 90–95%, respectively56). The paucity of anti-Tg and anti-TPO antibodies in those who develop autoimmune primary hypothyroidism implies a non-humoral pathway. As observed in chronic autoimmune thyroiditis, these hypothyroidism events may be secondary to: reduced number and/or function of immune regulatory suppressor cells (e.g. CD4+CD25+)57,58; the large diversity of the third complementarity-determining region of T-cell surface antigen receptors creating increased propensity of attacking thyroid tissue59; from Th1 cytotoxic T-cell apoptotic destruction of thyroid tissue60; or loss of self-tolerance61 by direct interaction of anti-PD1-/L1 agents with PD-L1/L2 expressed on normal thyroid tissue62. Since Th1 lymphocytes secrete interleukin-2, interferon gamma, and tumor necrosis factor-beta63, evaluation of these cytokines may help elucidate the role of these cells in anti-PD-1-induced non-humoral hypothyroidism.\n\nThis study has several limitations. This was a proof-of-concept study designed to screen for candidate autoantibody targets in a mixed solid tumor cohort. Our proteome was not comprehensive and may have missed potential targets during autoantibody candidate selection. During the candidate selection, the status of two patients changed due to the late development of significant toxicity events and four patients developed a toxicity event before the pre-cycle 3 blood draw, possibly skewing these preliminary results. Since the change in peptide quantity was used to select the candidates, the act of pooling serum samples among the four different patient groups may have diluted autoantibody targets present in low titers. Further, the candidates were selected based on peptide number increases from pre- to post-pembrolizumab, thus excluding possible candidates from baseline titers alone. Our study with mixed histologies, each in small numbers, creates a challenge in interpreting the CBR results. In total, 26 patients came off trial prior to pre-cycle 3 blood processing and were removed from the autoantibody analysis. This incomplete patient representation may bias event outcomes and distort the predictive/prognostic potential of the candidate autoantibodies. Since new irAEs from PD1 inhibition can occur many months after starting treatment43, and onset of hypothyroidism post-PD-1 initiation can range from 0.7 weeks to 19 months5, there is the possibility that ongoing patient follow up will yield new toxicity events. This is especially pertinent to the patients who developed non-thyroidal toxicity who have anti-thyroid antibodies present. The small number patients analyzed and limited number of response and toxicity events yields low statistical power and may give spurious results. Ultimately, our findings require further validation in an independent prospective dataset, with adequate sample size to detect histology-specific effects on survival.\n\n\nConclusions\n\nWe have shown an association of an increase in anti-Tg and anti-TPO titers between baseline at pre-cycle 3 pembrolizumab with toxicity, elevated pre-cycle 3 anti-Tg with toxicity, and elevated pre-cycle 3 anti-TPO with OS. However, anti-Tg and anti-TPO were unable to predict for non-thyroid toxicity. Future prospective trials evaluating these antibodies, ideally at a time point earlier than pre-cycle 3 of pembrolizumab or with other ICB agents, are needed to validate this finding. Enriching for patients with a single disease site will aid in determining their association with CBR. If successful, this will yield the first data for a minimally invasive, blood-based predictive biomarker to identify which patients derive benefit and/or toxicity from pembrolizumab, sparing unnecessary financial burden and delays to more appropriate care.\n\n\nData availability\n\nFigshare: Autoantibody dataset.xlsx. https://doi.org/10.6084/m9.figshare.1214959833.\n\nThis file contains the autoantibody dataset generated and assessed during this study, including patient outcomes, autoantibody titers and mass spectrometry data.\n\nFigshare: Extended data.docx. https://doi.org/10.6084/m9.figshare.12149601.v130.\n\nThis extended data file contains the following information:\n\nConcentration of protein in various tissue lysates used for autoantibody binding.\n\nAutoantibody immuno-mass spectrometry methods.\n\nImmunohistochemistry methods and MPS calculation.\n\nBeeswarm plots showing anti-Tg antibody titers in those with or without toxicity from pembrolizumab.\n\nStatistics of anti-Tg and anti-TPO antibody as continuous variables and their association with CBR and toxicity.\n\nBeeswarm plots showing anti-TPO antibody titers in patients with and without toxicity to pembrolizumab.\n\nBeeswarm plots showing anti-Tg antibody titers in those with or without CBR from pembrolizumab.\n\nBeeswarm plots showing anti-TPO antibody titers in those with or without CBR from pembrolizumab.\n\nKaplan-Meier PFS analysis using baseline and pre-cycle 3 anti-Tg.\n\nKaplan-Meier PFS analysis using baseline and pre-cycle 3 anti-TPO.\n\nKaplan-Meier analysis using Δ anti-Tg.\n\nKaplan-Meier analysis using Δ anti-TPO.\n\nAnalysis of covariate ethnicity with anti-Tg and anti-TPO titers as a categorical variable.\n\nAnalysis of covariate ethnicity with anti-Tg and anti-TPO titers as continuous variables.\n\nAnalysis of covariate gender with anti-Tg and anti-TPO titers as categorical variables.\n\nAnalysis of covariate gender with anti-Tg and anti-TPO titers as continuous variables.\n\nAnalysis of covariate PD-L1 MPS percentage as a categorical variable with anti-Tg and anti-TPO titers as categorical variables.\n\nAnalysis of covariate PD-L1 MPS percentage as a continuous variable with anti-Tg and anti-TPO titers as categorical variables.\n\nAnalysis of covariate PD-L1 MPS as a categorical variable and anti-Tg and anti-TPO titers as continuous variables.\n\nAnalysis of covariate age as a continuous variable with anti-Tg and anti-TPO titers as categorical variables.\n\nAnalysis of standard prognostic variables age and PD-L1 MPS with anti-Tg and anti-TPO titers, all as continuous variables.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Author contributions\n\n\n\nMI, MM, LLS and EPD wrote the paper. MM, AS, IB, IP and EPD conducted the experiments. MI, MM, LLS and EPD analyzed the data. MI, ARH, PLB, SL, AS, AAR and LLS are clinicians involved with enrolling patients. MP provided statistical support. All authors read the paper and concur with the submission. All authors read and approved the final manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the patients and their families for their participation.\n\n\nReferences\n\nFarkona S, Diamandis EP, Blasutig IM: Cancer immunotherapy: the beginning of the end of cancer? BMC Med. 2016; 14: 73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCully M: Combinations with checkpoint inhibitors at wavefront of cancer immunotherapy. Nat Rev Drug Discov. 2015; 14(6): 374–5. PubMed Abstract | Publisher Full Text\n\nSunshine J, Taube JM: PD-1/PD-L1 inhibitors. Curr Opin Pharmacol. 2015; 23: 32–8. 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}
|
[
{
"id": "69349",
"date": "07 Sep 2020",
"name": "Gerasimos Sykiotis",
"expertise": [
"Reviewer Expertise Thyroid pyhsiology",
"pathophysiology and disease."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript if fully well summarized in its abstract, hence there is no need to provide a separate summary here. I recommend to revise the text to address the following comments:\nThe main irAE in the cohort was hypothyroidism. It is then not surprising that the main autoantibodies identified were thyroid-specific proteins, and actually they were “the usual suspects”. In the Discussion, it is mentioned that new insights might be obtained by analyzing cohorts homogeneous as to the primary tumor site. But is there any evidence that the irAE profile depends on the primary tumor site? If so, then it should be cited in support. If not, then this statement should be revised. In either case, it may be more productive to profile (sub)cohorts homogeneous as to the type of irAE(s), rather than the primary tumor site.\n\nOn the one hand, the analyses shown in Table 2 show no association between anti-Tg antibody levels at baseline and toxicity. On the other hand, the first paragraph on page 9 mentions that hypothyroidism development during treatment was more common among patients with elevated anti-Tg titers at baseline. These two statements appear contradictory, and I would recommend to address and explain this directly in the Discussion. For example, is it because the “toxicity” assessed in Table 2 includes all irAEs?\n\nIn the second paragraph of page 9, it is mentioned that 60% of patients who did not develop hypothyroidism had elevated anti-Tg or anti-TPO antibody titers at baseline and/or pre-cycle 3. This % is quite high. I recommend that the authors mention specifically the % of patients with positive antibodies at baseline (this is actually done in the Discussion, but for the overall cohort, not specifically for the patients who did not develop hypothyroidism). Also, statistics aside, it appears intuitive to a clinician that if the % of positivity at baseline is so high among patients who do not develop hypothyroidism during treatment, then the predictive value of this baseline assessment will be very low for any given individual patient. I think that this merits a mention.\n\nI recommend to address whether patients with positive/elevated anti-Tg or anti-TPO antibodies at baseline were more likely to show an increase of these titers during treatment.\n\nWere any patients with pre-existing thyroid disease included in the discovery cohort (top of text in page 9)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "75094",
"date": "30 Nov 2020",
"name": "Francesco Salvatore",
"expertise": [
"Reviewer Expertise Reviewer Francesco Salvatore: pathophysiology and diseases",
"Reviewers Tommaso Porcelli and Domenico Salvatore: thyroid physiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this work by Music et al. was to identify biomarkers able to predict the clinical benefit and the risk of irAEs in patients treated with pembrolizumab. In a discovery analysis, the authors found anti-Tg Ab to be significantly increased by pembrolizumab. Therefore, anti-thyroid Ab (i.e., anti-Tg and anti-TPO Abs) were evaluated as predictive biomarkers of tumor response and treatment toxicity in 78 patients treated with pembrolizumab for different tumor types. The aim of the paper appears highly interesting. The authors clearly show that patients with an increase in baseline anti-Tg and anti-TPO titers after 7 weeks of treatment presented higher rate of toxicity in response to pembrolizumab. Moreover, the authors found an association between anti-TPO titer and overall survival, although without benefit in progression-free survival.\nInterestingly, 50% of patients that developed hypothyroidism as irAEs were treated with pembrolizumab for a melanoma cancer (i.e. 14% of enrolled patients). Moreover, as shown in table 1, the melanoma group presented the highest rate of patients with clinical benefit rate (CBR) (8/11, 73%) and with toxicity (6/11, 55%). Conversely, other cancers showed very different responses. For example, the triple negative breast cancer presented no cases of hypothyroidism, a low CBR (1/12, 8%) and a low toxicity (1/12, 8%). Accordingly, 6/11 of melanoma patients presented baseline thyroid-Ab alterations and 4/11 presented increase in thyroid-Ab after treatment. I would have suggested to comment in the discussion the strong association between anti-thyroid Ab and CBR/ toxicity in melanoma patients.\nThe cutoff to define auto-Ab elevation was set at 10.0 IU/ml. As shown in the “Autoantibody dataset” tables (ref 33), in 4 patients considered with elevated basal Tg-Ab, the Ab titer was <10.9 IU/ml (in one case was 10.06 IU/ml) and in 3 of these 4 patients there was no increase at pre-cycle 3 measurement. Similarly, 4 patients from the group with elevated pre-cycle 3 TPO-Ab presented only slightly elevated values (in 2 cases, 10.02 UI/ml). I’m not sure that values of 10.02 IU/ml can be considered as positive, especially if no increase (or a decrease) was observed after treatment. This is important because the reported association between overall survival and elevated pre-cycle 3 anti-TPO Ab is based on these values. I would have suggested to specify in the “Methods” section if serum anti-Ab measurement were repeated for confirmation and if the reported values are a mean value of more measurements.\nIn Figure 1, in the “outcomes” boxes, the reasons for stopping treatment include both clinical progression and RECIST progression. I would have specified in the “Methods” section which are the criteria for the definition of “clinical progression” and when/why this parameter is chosen instead of RECIST progression to decide for the treatment stop.\nIn conclusion this is an highly interesting paper, the topic of this study is interesting and the work provides new insight into the field. This proof of concept paper has the additional merit to allow other cohorts of patients to be enrolled and therefore extend the casistics.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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
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https://f1000research.com/articles/9-337
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https://f1000research.com/articles/8-1841/v1
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01 Nov 19
|
{
"type": "Research Article",
"title": "Do north-eastern German pharmacies recommend a necessary medical consultation for acute diarrhoea? Magnitude and determinants using a simulated patient approach",
"authors": [
"Bernhard Langer",
"Christian Kunow",
"Christian Kunow"
],
"abstract": "Background: To determine if pharmacy staff in Germany referred patients to a medical consultation for a scenario in which consulting a doctor was mandatory (‘appropriate outcome’) and what the quality of questioning and – if a medication was dispensed – the quality of information provided were in this context. Moreover, to determine which factors predicted a necessary referral to a doctor. Methods: A cross-sectional covert simulated patient study was conducted in a random sample of community pharmacies stratified by location in the German state of Mecklenburg-Vorpommern. Each pharmacy was visited once by one of four trained investigators. They simulated a symptom-based request involving a grandmother with acute diarrhoea. A multivariate binary logistic regression analysis using potential variables from bivariate analysis was carried out to determine the predictors for a referral to a doctor. Results: All 199 planned visits were conducted. A necessary referral to a doctor was recommended in 59.8% (n=119) of all test purchases. Multivariate analysis revealed that a non-pharmacist as opposed to a pharmacist and two or more questions as opposed to no questions were significantly associated with a referral to a doctor. Conclusions: Regarding the necessary referral to a doctor, an enormous potential for improvement was revealed for community pharmacies in the German state of Mecklenburg-Vorpommern. The results should make both the Mecklenburg-Vorpommern pharmacy association and legislators aware of the need to significantly escalate their quality management efforts.",
"keywords": [
"Non-prescription drugs",
"Community pharmacies",
"Consultation",
"Patient simulation",
"Diarrhoea",
"Germany"
],
"content": "Introduction\n\nAcute diarrhoea is one of the most common diseases worldwide1, including in Germany2. While worldwide about 1 billion people develop acute diarrhoea each year2, causing about 2 billion cases of acute diarrhoea3, about 30% of the German population suffer from acute diarrhoea episodes each year2. Because those affected report becoming ill on average 1.7 times per year2, this means that about 42 million cases of acute diarrhoea can therefore be expected in Germany.\n\nAccording to the guidelines of the German Society of General Medicine and Family Medicine, acute diarrhoea is an imbalance between secretion and absorption in the bowels, whereby the symptoms last fewer than 14 days and are associated with an increased frequency (≥ three loose stools in a day) or an increased water content (≥ 75%) or an increased stool weight (≥ 250 g)4.\n\nAcute diarrhoea may be a result of infections with viruses (for example, noroviruses or rotaviruses), bacteria (for example, non-typhoidal Salmonella or diarrheagenic Escherichia coli) or parasites (for example, Cryptosporidium parvum)5. More than 90% of cases result from infections, with viral infections being the most common cause6. Bacterial infections on the other hand are more often associated with travel, food poisoning and comorbidities. Medications (e.g. antibiotics, cytostatics, diuretics), gastroenterologic disorders (e.g. ulcerative colitis, Crohn’s disease), endocrine disorders (e.g. hyperthyroidism, adrenocortical insufficiency) or other medical conditions (e.g. amyloidosis) can also trigger acute diarrhoea5.\n\nIn Germany there is no medical consultation in about two-thirds of cases of acute diarrhoea2. Therefore, advice should be provided by the pharmacy, not least because of the wide range of possible causes, when the diarrhoea is self-medicated with over-the-counter (OTC) medications only available from pharmacies in Germany. As part of a high quality consultation, pharmacy staff should also identify when it is necessary to refer a patient to a medical consultation based on the symptoms7,8. In Germany, in addition to pharmacists, pharmacy technicians and pharmaceutical technical assistants are also permitted to advise on and sell medications but only under the supervision of pharmacists.\n\nAlthough studies available from Germany regarding the quality of advice provided in pharmacies reveal significant deficits, analogous to the international literature9–12, indications other than acute diarrhoea in adults are usually investigated in scientific13,14 and non-scientific studies15–19. The quality of advice provided for acute diarrhoea in adults was analysed for Germany for the first time in 2014 and also identified clear deficiencies20. Despite performance feedback used in the process, a follow-up study identical to that conducted in 2014 was carried out for the first time in Germany in 2017 and also showed poor quality of advice21,22. All the above mentioned studies have in common that they are based on the simulated patient (SP) method to determine the quality of advice provided. In this method test persons simulate participation in an apparently real service provision process to subsequently evaluate the process and reveal any deficiencies as a result23.\n\nIn the baseline study in 2014 (as well as the follow-up study in 2017) the four scenarios investigated were designed as ‘moderate’ in that none of the scenarios predicted referral to a medical consultation by the pharmacy staff as an ‘appropriate outcome’. Both studies also investigated only the quality of advice in 21 pharmacies in a medium-sized northern German city (a total of 84 test purchases for each study).\n\nThe aim of this study was, therefore, to determine on the basis of a considerably larger number of pharmacies whether the pharmacy staff referred patients to a medical consultation for a scenario in which consulting a doctor was mandatory (‘appropriate outcome’) and what the quality of questioning and – if a medication was dispensed – the quality of information provided were in this context. Factors influencing the ‘appropriate outcome’ were also determined analogous to other national and international studies14,21,24–27.\n\n\nMethods\n\nA cross-sectional study design was chosen in accordance with the ‘STROBE Statement – Checklist of items that should be included in reports of cross-sectional studies’28 and, to determine the quality of advice provided in the pharmacies investigated, based on the highly recommended29 SP method that is often used internationally23,30–33 as a form of participatory observation34.\n\nBecause of time constraints of the SPs, the test purchases took place over winter between 1 November and 15 December 2018 in the German state of Mecklenburg-Vorpommern (31 December 2017: approx. 1.61 million residents, 23,214 km2 area, low population density of 69.4 residents/km2)35. Upon request by telephone, the Pharmacy Association of Mecklenburg-Vorpommern did not want to provide a list of all registered community pharmacies in the state. Consequently, these pharmacies were identified using the pharmacy finder of the website Apotheken-Umschau.de36. All community pharmacies that had a postcode in the state of Mecklenburg-Vorpommern on the reference date of 1 October 2018 using the postcode search of the pharmacy finder were included in the study. These hits were validated with a corresponding Google search. As a result, a basic population of N=396 pharmacies was determined. A comparison with the last available information from the German Federal Chamber of Pharmacies (ABDA) for the end of 2018 regarding the total number of pharmacies in Mecklenburg-Vorpommern37 showed a 99% agreement. The minimum necessary sample size (n) was determined for a population size (N) of 396 and an error margin (e) of 0.05 using the following formula, which is based on a degree of variability of P=0.5 and a 95% confidence interval38:\n\n\n\nIn Germany there are no studies yet on a necessary referral to a doctor for acute diarrhoea by community pharmacies. Therefore, the degree of variability is unknown. The assumed degree of variability of P=0.5 maximises the required sample size. The 396 pharmacies were stratified by location of the pharmacy as an indicator for urban/rural and assigned a random number using the MS Excel random number generator and then a simple random sampling was performed in each stratum to select the 199 participating pharmacies.\n\nThe Ordinance on the Operation of Pharmacies in Germany includes an obligation for pharmacies to introduce a quality management system. The aim is to ensure a preferably adequate advice outcome39. To ensure that this can be achieved, the Federal Chamber of Pharmacies has drafted various guidelines and tools, including the tool ‘Information and advice as part of self-medication using the example of self-diagnosis of diarrhoea’40. This forms the basis of the symptom-based test scenario developed (Table 1) and the evaluation form used (see the assessment items in Table 4). The recommendation to consult a doctor by pharmacy staff is defined as the appropriate outcome and the test scenario was also designed according to this. Diarrhoea being present for five days as well as a high fever (40°C) are both considered to exceed the limits of self-medication according to the tool. The evaluation form includes a total of eleven items with the first six items evaluating whether appropriate questions were asked. On this basis the pharmacy staff should then decide whether to recommend that the patient consult a doctor (item seven). Because the tool also considers ‘dispensing a medication in an appropriate quantity up to referring to a doctor’ within the discretionary powers of the pharmacy staff, also in the case of the limits of self-medication being exceeded and the necessary referral to consult a doctor, items eight to eleven evaluate whether a medication was dispensed and whether in the process information on the dosage, duration and side effects was provided.\n\nOnly objective items were used in order to avoid a subjective assessment and thus latitude in the evaluation by the SPs (for example, on the friendliness of the pharmacy staff). Therefore, to complete the individual items only dichotomous scales were used (closed yes/no questions). To avoid the Hawthorne effect41 and to ensure a realistic consultation situation, the test purchases took place without first informing the pharmacies included in the random sample in accordance with other national20,21 and international26,42,43 studies.\n\nAs part of their three-semester research project at the Neubrandenburg University of Applied Sciences, two female and two male Masters students in the Health Sciences faculty, that is, four SPs, were available for the test purchases. Each pharmacy was visited once (a total of 199 test purchases), whereby the pharmacies were distributed randomly across the four SPs (three SPs 50 test purchases, one SP 49 test purchases).\n\nBefore the start of the data collection, each of the SPs familiarised themselves with the theoretical principles of the methodology and the contents of the evaluation form.\n\nA pilot study with four test purchases was then carried out by each of the SPs to train the SPs in the use of the methodology and to verify the functionality of the collection form and the four test scenarios. The total 16 test purchases were carried out in pharmacies that were not part of the stratified random sample. No changes to the test scenarios and the collection form were required after testing the scenarios.\n\nThe test purchases were carried out on different days of the week and at different times of the day. The SPs made their request to the pharmacy staff who first approached them. The SPs only provided additional information if they were then asked by the pharmacy staff to ensure that the information provided is invariable.\n\nAlong with the items on the evaluation form, the SPs planned, analogous to the international literature (Table 2), to also collect a number of variables before, during and after the test purchases that may possibly affect the appropriate outcome.\n\nNote: The possible influencing factors are taken from the particular literature sources.\n\nThe particular evaluation form was completed immediately after the test purchases by the SPs to minimise any recall bias of the study results due to faulty memories.\n\nData were entered in duplicate into and analysed with SPSS software (IBM, Armonk, NY, USA), version 25 for Windows. As part of the descriptive statistics, frequencies and percentages were determined. Furthermore, 95% confidence intervals (CI) for categorical data using bootstrapping methods were also reported. Because the rule of thumb for sample size assumptions (minimum of 10 events per predictor variable) is given for a logistical regression45, a binary logistic regression model was developed to identify the influence of various independent variables (Table 2) on the appropriate outcome. All independent variables were checked for outliers and multicollinearity. According to Hosmer et al.46, variables with a P value less than 0.25 in the bivariate analysis were included in the multivariate analysis. Crude odds ratios (COR) and adjusted odds ratios (AOR), 95% confidence intervals (CI) and P values were reported. A P value less than 0.05 was deemed significant in multivariate analysis.\n\nThe study protocol was approved by the institutional ethics committee of the Neubrandenburg University of Applied Sciences (registration number: HSNB/GPM/139/18). According to the ‘Guideline for the use of mystery research in market and social research’47, the data collected were anonymised and recorded in such a way that the pharmacies involved could not be identified. Because of the nature of this study, pharmacies were not asked for consent to participate. Recruited students provided their written informed consent to act as SPs.\n\n\nResults\n\nAll 199 planned test purchases were actually carried out, with a total of €784.70 used from the primary author’s own resources. Table 3 shows the socio-demographic data for the 199 pharmacies and the advising pharmacy staff.\n\nThis reveals that the pharmacies are mostly in locations with competing pharmacies nearby. In addition, only a minority of the pharmacies had a quality certificate. In most of the test purchases, the advising pharmacy staff were female, between 30 and 49 years of age and did not have a pharmacy degree (non-pharmacist).\n\nThe appropriate outcome was achieved in 59.8% (n=119) of all test purchases (Table 4). Most commonly the question ‘for whom is the medication?’ was asked (75.4%, n=150), while ‘clarification by a doctor’ was asked the least (17.6%, n=35). In 87.9% (n=175) of all test purchases a medication was dispensed. Regarding the test purchases in which a medication was dispensed, in 88.6% (n=155) information was provided about the dosage of the medication whereas information about possible side effects was only provided by the pharmacy staff in 8.0% (n=14) of test purchases.\n\nThe binary logistic regression model is shown in Table 5. Bivariate analysis demonstrated that only three (quality certificate, professional group of the pharmacy staff, number of questions asked) of eight predictor variables having a P value < 0.25 were included in a multivariate logistic regression model.\n\nAbbreviations: COR = Crude Odds Ratio; AOR = Adjusted Odds Ratio.\n\n* significant at P < 0.05.\n\nIn this multivariate analysis, being a non-pharmacist (AOR = 2.446; 95% CI = 1.025–5.835; p = 0.044) or pharmacy staff with an undetermined professional group (AOR = 3.269; 95% CI = 1.208–8.843; p = 0.020) as opposed to a pharmacist and two (AOR = 15.291; 95% CI = 4.876–47.955; p < 0.001) or three or more (AOR = 23.406; 95% CI = 8.204–66.773; p < 0.001) questions as opposed to no questioning were significantly associated with referral to a doctor. The location of the pharmacy, the presence of a quality certificate and a queue, the age and the gender of the pharmacy staff, as well as the time of day of the test purchases did not have any significant effect on the appropriate outcome. The model returned a Nagelkerke R2 value of 0.45.\n\n\nDiscussion\n\nAlthough the appropriate outcome, that is, the recommendation to consult a doctor, was achieved in approximately 60% of all test purchases, approximately 40% of all pharmacies did not recommend that the patient consult a doctor even though this would have been necessary. Due to the design of the scenario as a symptom-based query, the study results obtained would probably be worse in everyday consultations with its mix of symptom- and product-based queries because in the international and national literature (otherwise identical) scenarios showed a better quality of advice for a symptom-based query than for a medication-based query20,48. In another study a recommendation rate for a necessary medical consultation of 57% was determined for a medication-based query, likewise for acute diarrhoea in adults, in Australian community pharmacies27. Considerably worse results for the recommendation of a necessary medical consultation are seen in the international literature for other indications such as pain (recommendation rate: approx. 27%43), vaginal thrush (recommendation rates: 10% and 22%49) and asthma (recommendation rates: 40% and 19%50) which were all medication-based queries. In contrast, the only study that has investigated the recommendation for a necessary medical consultation to date in Germany showed a very high recommendation rate of 90% once again for a medication-based query for an antacid13.\n\nPossible reasons discussed in the international literature for the poor quality of advice provided include lack of time, manpower, interest and knowledge on behalf of the pharmacy staff10,48,51,52. In regards to the lack of knowledge, as part of the pharmacy degree (pharmacist) or professional training (pharmaceutical technical assistant) patient consultations should be more intensively trained using examples44. In addition, the systematic use of checklists could also help to ensure a better quality of advice in daily patient contact53.\n\nOn the other hand, the international literature reveals that pharmacy staff most definitely have the knowledge to provide good advice, but it is only inadequately applied in actual patient contact51,54–56.\n\nIn a German study, pharmacy staff suggested that this is due to the worry that the client may feel patronised13. In contrast to this, another German study determined that most patients would like to have a consultation in the pharmacy; however, due to the study design this involved rather health-conscious and therefore not representative study participants57. In a very recent German study it was stated by pharmacy staff that patients frequently did not want any advice in terms of drug handling, sometimes due to a lack of time58. In a somewhat older English study, 62.5% of the patients interviewed did not expect to be asked any questions by the pharmacy staff during their last purchase of a non-prescription medication59. Similar results were obtained in a more recent study in Qatar in which the pharmacist cited ‘no interest by the patients’ as the central reason for providing insufficient advice42. Another very recent qualitative study from Australia was also able to determine in this context that from the perspective of both the consumers and the pharmacy staff, the frequent lack of privacy is also an important reason for the poor exchange of information60. It would, therefore, be necessary to require pharmacy owners in future to provide appropriate spaces that ensure privacy.\n\nAnalogous to other international studies50,61,62, the number of questions asked had a significant effect on the appropriate outcome. This is based on the rationale that the right decision – in our case to recommend consulting a doctor – can only be made if appropriate questions are asked and thus information is obtained about the patient. In contrast to this, a Saudi Arabian study revealed that there was no relationship between the number of questions asked and the appropriate outcome – in this case the dispensing of prescription medications without a medical prescription25. However, the outcome ‘dispensing prescription medications without a medical prescription’ is not comparable to the outcome ‘recommendation to consult a doctor’. While for the first a sensible decision can be made even without asking appropriate questions (that is, refusing to dispense a prescription medication without a corresponding medical prescription), for the latter a sensible decision is only possible on the basis of appropriately asked questions.\n\nIn regards to individual questions, the question about the duration of the acute diarrhoea was asked more frequently compared to comparable national studies20,21. In a recent SP study, also for acute diarrhoea in adults, that was conducted in Iraq, this question was asked in almost 80% of all test purchases63. Considerably worse, on the other hand, are the results of a comparable Turkish SP study, which also investigated the quality of advice provided for acute diarrhoea in adults, in which this question was only asked in 26% of all test purchases64. Because certain medications can cause diarrhoea, the question about any pre-existing medical conditions or the patient’s medical history is particularly important65. This question was asked in this study in approximately one-third of the test purchases and is thus more frequent than in the comparable national studies20,21. In the similar Turkish SP study, this question was not asked in a single test purchase64.\n\nDepending on the particular criteria being investigated, there are also clear differences in the information that is given to the patients when medication is dispensed. Analogous to the national20,21 and international literature54,66, information is often provided about the dosage of the medication but rarely about possible side effects. An active query by patients could help to greatly improve the frequency of advice provided for the criterion ‘side effects’, as was shown in a recent SP study from Tanzania67.\n\nAlong with the number of questions asked, the professional group of the pharmacy staff providing the advice also had a significant influence on the appropriate outcome. Non-pharmacists (pharmaceutical technical assistants and pharmacy technicians) have a significantly higher likelihood of achieving the appropriate outcome than pharmacists. On the other hand, two comparable SP studies from Germany and Australia – also for acute diarrhoea in adults – did not show any significant difference in the quality of advice provided by the two professional groups21 or a significantly higher likelihood for pharmacists27 of achieving the appropriate outcome. In the Turkish SP study pharmacists made significantly more recommendations to their patients than pharmaceutical technical assistants64. For indications other than acute diarrhoea in adults there was an analogous picture – either a significantly better quality of advice provided by pharmacists compared to non-pharmacists14,62 or no significant differences between the two professional groups48,54,68. Due to inconsistent national and international studies and in light of the surprising results obtained in this study, future studies should investigate in greater depth this influencing factor for the advice provided.\n\nThis is the first German study that investigated to what degree community pharmacies recommend a necessary medical consultation for acute diarrhoea in adults. The sample size used in the study (n=199) is also considerably larger than the international median sample size (n=112) for SP studies69. An SP study design also avoids social desirability, unlike a survey. By using 4 SPs, the study also satisfies the requirement of not having fewer than 2 (generalisable) but also not too many (standardisable) SPs69.\n\nBy way of qualification, it must be stated that the results only refer to one scenario. In addition, only one indication was investigated.\n\nBecause the study results only refer to one German state, future studies should be expanded to include additional or all German states.\n\nThe results also refer only to a quite specific time because the study is a cross-sectional study. There is further need for research to determine whether and, if yes, how the results change over time, which is only possible with a longitudinal study design.\n\nThe audio recordings recommended in the literature for quality assurance70 must be omitted for data privacy reasons because all pharmacies would have to be informed about this in advance, which would jeopardise the covert study design. Although the particular evaluation forms were filled out by the SPs immediately after visiting the pharmacies and only objective evaluation criteria were used, recall bias26 and the intra- and inter-observer variabilities typical for SP studies71 cannot be completely ruled out. For greater quality assurance, future SP studies could always carry out and evaluate test purchases in parallel using two persons (1 SP and 1 observer)72.\n\nAlthough in the literature it is recommended for reasons of effectiveness to provide performance feedback to initiate improvement measures immediately after the particular test purchase23, this was omitted because the student SPs would probably not be accepted by pharmacy staff as briefing partners14. The restrictions on time and resources and the very high number of pharmacies visited mean that it was also not possible to provide the pharmacies with individual written performance feedback after the test purchases.\n\n\nConclusions\n\nIn terms of achieving the appropriate outcome, an enormous potential for improvement was identified for community pharmacies in the German state of Mecklenburg-Vorpommern. Furthermore, more questioning and involvement by non-pharmacists was identified as relevant factors influencing the appropriate outcome. There is a further need for research to identify why the advice provided is so often poor and particularly why the quality of advice provided by pharmacists is so inadequate. The results should make both the Mecklenburg-Vorpommern pharmacy association and legislators aware of the need to significantly escalate their quality management efforts. In this context appropriate mandatory continuing education courses as well as regular independent reviews with an adequate sanction mechanism could provide a stimulus to sustainably improve the quality of advice73,74.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Do north-eastern German pharmacies recommend a necessary medical consultation for acute diarrhoea? Magnitude and determinants using a simulated patient approach, https://doi.org/10.7910/DVN/5KVLW475\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nThe authors thank the four master students for acting as simulated patients.\n\n\nReferences\n\nGBD 2017 Disease and Injury Incidence and Prevalence Collaborators: Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018; 392(10159): 1789–1858. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLankisch PG, Mahlke R, Lübbers H, et al.: Zertifizierte medizinische Fortbildung: Leitsymptom Diarrhö. Dtsch Arztebl. 2006; 103(5): A261–A269. Reference Source\n\nFarthing M, Salam MA, Lindberg G, et al.: Acute diarrhea in adults and children: a global perspective. J Clin Gastroenterol. 2013; 47(1): 12–20. PubMed Abstract | Publisher Full Text\n\nSander M, Gerlach K: S1-Leitlinie Akuter Durchfall. Deutsche Gesellschaft für Allgemeinmedizin und Familienmedizin. 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PubMed Abstract | Publisher Full Text\n\nKashour TS, Joury A, Alotaibi AM: Quality of assessment and counselling offered by community pharmacists and medication sale without prescription to patients presenting with acute cardiac symptoms: a simulated client study. Eur J Clin Pharmacol. 2016; 72(3): 321–328. PubMed Abstract | Publisher Full Text\n\nSaba M, Diep J, Bittoun R, et al.: Provision of smoking cessation services in Australian community pharmacies: a simulated patient study. Int J Clin Pharm. 2014; 36(3): 604–614. PubMed Abstract | Publisher Full Text\n\nCollins JC, Schneider CR, Faraj R, et al.: Management of common ailments requiring referral in the pharmacy: a mystery shopping intervention study. Int J Clin Pharm. 2017; 39(4): 697–703. PubMed Abstract | Publisher Full Text\n\nSTROBE Statement – Checklist of items that should be included in reports of cross-sectional studies. (accessed 08 February 2019). 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nParavattil B, Kheir N, Yousif A: Utilization of simulated patients to assess diabetes and asthma counseling practices among community pharmacists in Qatar. Int J Clin Pharm. 2017; 39(4): 759–768. PubMed Abstract | Publisher Full Text\n\nByrne GA, Wood PJ, Spark MJ: Non-prescription supply of combination analgesics containing codeine in community pharmacy: A simulated patient study. Res Social Adm Pharm. 2018; 14(1): 96–105. PubMed Abstract | Publisher Full Text\n\nLanger B, Bull E, Burgsthaler T, et al.: [Using the simulated patient methodology to assess counselling for acute diarrhoea - evidence from Germany]. Z Evid Fortbild Qual Gesundhwes. 2016; 112: 19–26. PubMed Abstract | Publisher Full Text\n\nPeduzzi P, Concato J, Kemper E, et al.: A simulation study of the number of events per variable in logistic regression analysis. J Clin Epidemiol. 1996; 49(12): 1373–1379. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchneider CR, Everett AW, Geelhoed E, et al.: Intern pharmacists as change agents to improve the practice of nonprescription medication supply: Provision of salbutamol to patients with asthma. Ann Pharmacother. 2010; 44(7–8): 1319–1326. PubMed Abstract | Publisher Full Text\n\nSurur AS, Getachew E, Teressa E, et al.: Self-reported and actual involvement of community pharmacists in patient counseling: a cross-sectional and simulated patient study in Gondar, Ethiopia. Pharm Pract (Granada). 2017; 15(1): 890. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAyalew M: Medication Counseling Practice in Ethiopia: A Systematic Review. J Basic Clin Pharma. 2017; 8: S001–S005. Reference Source\n\nChong WW, Aslani P, Chen TF: Adherence to antidepressant medications: an evaluation of community pharmacists’ counseling practices. Patient Prefer Adherence. 2013; 7: 813–825. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGokcekus L, Toklu HZ, Demirdamar R, et al.: Dispensing practice in the community pharmacies in the Turkish Republic of Northern Cyprus. Int J Clin Pharm. 2012; 34(2): 312–324. PubMed Abstract | Publisher Full Text\n\nOgbo PU, Aina BA, Aderemi-Williams RI: Management of acute diarrhea in children by community pharmacists in Lagos, Nigeria. Pharm Pract (Granada). 2014; 12(1): 376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrata C, Marjadi B, Schneider CR, et al.: Information-gathering for self-medication via Eastern Indonesian community pharmacies: a cross-sectional study. BMC Health Serv Res. 2015; 15: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnwand T, Schaefer M: Umfrage. Individuelle Beratung statt kommentarloser Abgabe. Pharm Ztg. 2002; 147: 114–116. Reference Source\n\nSchumacher PM, Neininger MP, Kaune A, et al.: Counseling patients on correct drug handling in German community pharmacies: experiences and opinions of pharmaceutical staff. Int J Clin Pharm. 2019; 41(1): 151–158. PubMed Abstract | Publisher Full Text\n\nMorris CJ, Cantrill JA, Weiss MC: “One simple question should be enough”: consumers' perceptions of pharmacy protocols. Int J Pharm Pract. 1997; 5(2): 64–71. Publisher Full Text\n\nSeubert LJ, Whitelaw K, Boeni F, et al.: Barriers and Facilitators for Information Exchange during Over-The-Counter Consultations in Community Pharmacy: A Focus Group Study. Pharmacy (Basel). 2017; 5(4): 65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollins JC, Schneider CR, Naughtin CL, et al.: Mystery shopping and coaching as a form of audit and feedback to improve community pharmacy management of non-prescription medicine requests: an intervention study. BMJ Open. 2017; 7(12): e019462. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatson MC, Bond CM, Grimshaw J, et al.: Factors predicting the guideline compliant supply (or non-supply) of non-prescription medicines in the community pharmacy setting. Qual Saf Health Care. 2006; 15(1): 53–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIbrahim IR, Palaian S, Ibrahim MI: Assessment of diarrhea treatment and counseling in community pharmacies in Baghdad, Iraq: A simulated patient study. Pharm Pract (Granada). 2018; 16(4): 1313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSancar M, Tezcan E, Okuyan B, et al.: Assessment of the Attitude of Community Pharmacists and Pharmacy Technicians towards Diarrhea: A Simulated Patient Study in Turkey. Trop J Pharm Res. 2015; 14(8): 1509–1515. Publisher Full Text\n\nAbraham B, Sellin JH: Drug-induced diarrhea. Curr Gastroenterol Rep. 2007; 9(5): 365–372. PubMed Abstract | Publisher Full Text\n\nIbrahim MI, Palaian S, Al-Sulaiti F, et al.: Evaluating community pharmacy practice in Qatar using simulated patient method:acute gastroenteritis management. Pharm Pract (Granada). 2016; 14(4): 800. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHorumpende PG, Sonda TB, van Zwetselaar M, et al.: Prescription and non-prescription antibiotic dispensing practices in part I and part II pharmacies in Moshi Municipality, Kilimanjaro Region in Tanzania: A simulated clients approach. PLoS One. 2018; 13(11): e0207465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKayshap KC, Nissen LM, Smith SS, et al.: Management of over-the-counter insomnia complaints in Australian community pharmacies: a standardised patient study. Int J Pharm Pract. 2014; 22(2): 125–134. PubMed Abstract | Publisher Full Text\n\nWatson MC, Norris P, Granas AG: A systematic review of the use of simulated patients and pharmacy practice research. Int J Pharm Pract. 2006; 14(2): 83–93. Publisher Full Text\n\nWerner JB, Benrimoj SI: Audio Taping Simulated Patient Encounters in Community Pharmacy to Enhance the Reliability of Assessments. Am J Pharm Educ. 2008; 72(6): 136. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBardage C, Westerlund T, Barzi S, et al.: Non-prescription medicines for pain and fever--A comparison of recommendations and counseling from staff in pharmacy and general sales stores. Health Policy. 2013; 110(1): 76–83. PubMed Abstract | Publisher Full Text\n\nMahmoud AA, Althobaiti OA, Althobaiti AE, et al.: Community pharmacists' response to acute back pain: A simulated patient study in western region, Saudi Arabia. GSC Biological and Pharmaceutical Sciences. 2018; 2(2): 25–32. Publisher Full Text\n\nChowdhury F, Sturm-Ramirez K, Mamun AA, et al.: Effectiveness of an educational intervention to improve antibiotic dispensing practices for acute respiratory illness among drug sellers in pharmacies, a pilot study in Bangladesh. BMC Health Serv Res. 2018; 18(1): 676. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdalat A: Pro und Kontra: Pflichtfortbildung für Apotheker? DAZ.ONLINE vom 12.07.2018. (accessed 22 September 2018). Reference Source\n\nLanger B, Kunow C: \"Replication Data for: Do north-eastern German pharmacies recommend a necessary medical consultation for acute diarrhoea? Magnitude and determinants using a simulated patient approach.\"2019. Harvard Dataverse, V2, UNF: 6:ThCfNrfKdA/eRzJFf1L7yQ== [fileUNF] http://www.doi.org/10.7910/DVN/5KVLW4"
}
|
[
{
"id": "56075",
"date": "15 Nov 2019",
"name": "Mohamed Izham Mohamed Ibrahim",
"expertise": [
"Reviewer Expertise Social-behavioral and Administrative aspects of health and pharmacy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me to review this article. It is an interesting study using an interesting approach i.e. SP. I have a few comments:\nThe conclusion in the abstract section must be consistent with the objective of the study and main findings.\n\nPercentages reported in tables must equal to 100% - kindly check all figures.\n\nTables cited in the main text should in proper order.\n\nEven though due to the nature of the study, subjects selected were not asked for consent, did the authors contact them after the study informing that a study was conducted with reasons? This is one way that the researchers could resolve the issue of informed consent prior to the study.\n\nCreate a paragraph on Study recommendations at the end of the Discussion section. Consider to move some parts of the conclusion. Rewrite the conclusion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "5447",
"date": "27 Apr 2020",
"name": "Bernhard Langer",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our manuscript and also for the very helpful and detailed comments, recommendations and questions. See our point-by-point response to the reviewer’s comments below. Thank you for allowing me to review this article. It is an interesting study using an interesting approach i.e. SP. I have a few comments: The conclusion in the abstract section must be consistent with the objective of the study and main findings. Thank you for this comment. The conclusion has been restructured to be consistent with the objectives of the study and main findings. Percentages reported in tables must equal to 100% - kindly check all figures. Thank you for this comment. We have checked all percentages reported in tables and made corrections if necessary. Tables cited in the main text should in proper order. Thank you for this comment. We have ensured that the tables are cited in the correct order. Even though due to the nature of the study, subjects selected were not asked for consent, did the authors contact them after the study informing that a study was conducted with reasons? This is one way that the researchers could resolve the issue of informed consent prior to the study. Thank you for this comment. Yes, we contacted the subjects after the study informing them that an SP study was conducted with reasons. Therefore, the following sentence and two additional references have been added: ‘To resolve the issue of informed consent, the authors contacted the pharmacies by mail and email after the study informing them that an SP study had been conducted with the corresponding background information 48, 49 .’ Create a paragraph on Study recommendations at the end of the Discussion section. Consider to move some parts of the conclusion. Rewrite the conclusion section. Thank you for this comment. We have created a new paragraph on study recommendations at the end of the discussion section and have moved some parts of the conclusion to this new paragraph. Additionally, the conclusions section has been rewritten."
}
]
},
{
"id": "59628",
"date": "10 Feb 2020",
"name": "Suleiman I. Sharif",
"expertise": [
"Reviewer Expertise Drug Utilization Studies",
"Pharmacy education",
"Pharmacy Practice",
"Neuropharmacology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 thank you for trusting me with the review of this article which presents a well-designed and executed study. I do approve of indexing the article after some answers and minor corrections to be provided by the authors.\nEthical approval: I appreciate that in this study obtaining a written consent from the staff of the pharmacies would have greatly and negatively impacted the results and any “appropriate outcome” obtained would have been a biased one.\n\nPre-piloting the scenario with other than the researchers may have improved the approach.\n\nThe SPs made their request to the pharmacy staff who first approached them. Were the SPs certain whether “the pharmacy staff who first approached them” a pharmacist or pharmacy technician? In table 3, about 72% were not pharmacist and not able to determine. Moreover, the authors stated in the results that “In most of the test purchases, the advising pharmacy staff were females, between 30 and 49 years of age and did not have a pharmacy degree (non-pharmacist)”. Since those they should practice under the supervision of a pharmacist, the authors need to elaborate on this in relation to their discussion where they state” Non-pharmacists (pharmaceutical technical assistants and pharmacy technicians) have a significantly higher likelihood of achieving the appropriate outcome than pharmacists”.\n\nPoint 2 must be also discussed in relation to their conclusion where they state” Furthermore, more questioning and involvement by non-pharmacists was identified as relevant factors influencing the appropriate outcome. There is a further need for research to identify why the advice provided is so often poor and particularly why the quality of advice provided by pharmacists is so inadequate”.\n\nIn table 5, under Queue and Number of questions asked the percentages do not total to 100%. Therefore, the authors need to check their original data, or declare “Missing data” in the table to total the percentage to 100%.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5448",
"date": "27 Apr 2020",
"name": "Bernhard Langer",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our manuscript and also for the very helpful and detailed comments, recommendations and questions. See our point-by-point response to the reviewer’s comments below. I thank you for trusting me with the review of this article which presents a well-designed and executed study. I do approve of indexing the article after some answers and minor corrections to be provided by the authors. Ethical approval: I appreciate that in this study obtaining a written consent from the staff of the pharmacies would have greatly and negatively impacted the results and any “appropriate outcome” obtained would have been a biased one. Thank you for this comment. Yes, that is why we did not ask for consent to participate in advance. To clarify, the relevant sentence has been restructured as follows: ‘CPs were not asked for consent to participate prior to the study being conducted because obtaining written consent would have significantly and negatively impacted the results.’ Additionally, the following sentence was added: ‘To resolve the issue of informed consent, the authors contacted the CPs by mail and email after the study informing them that an SP study had been conducted with the corresponding background information 48, 49 .’ Pre-piloting the scenario with other than the researchers may have improved the approach. Thank you for this comment. Therefore, the following sentences and an additional reference have been added to the limitations: ‘Conducting the pilot study with persons other than the researchers may have improved the approach 73 , because the perspective of persons not involved in developing the study design would have been considered. On the other hand, carrying out the pilot study also aimed to train the SPs in the use of the methodology, which again would no longer have been possible.’ The SPs made their request to the pharmacy staff who first approached them. Were the SPs certain whether “the pharmacy staff who first approached them” a pharmacist or pharmacy technician? Thank you for this comment. This information was given in Table 2: ‘Exact measurement based on the name tag, the receipt and, if necessary, using a telephone query by the SP after completing the test purchase.’ Despite these efforts, the professional group of the pharmacy staff could not be determined in 55 of 199 test purchases. In table 3, about 72% were not pharmacist and not able to determine. Moreover, the authors stated in the results that “In most of the test purchases, the advising pharmacy staff were females, between 30 and 49 years of age and did not have a pharmacy degree (non-pharmacist)”. Since those they should practice under the supervision of a pharmacist, the authors need to elaborate on this in relation to their discussion where they state ‘Non-pharmacists (pharmaceutical technical assistants and pharmacy technicians) have a significantly higher likelihood of achieving the appropriate outcome than pharmacists.’ Thank you for this comment. We have expressed ourselves misleadingly. Therefore, we have deleted ‘but only under the supervision of pharmacists’ in the introduction. To better explain the word ‘non-pharmacist’, we have additionally restructured the sentence as follows: ‘In Germany, non-pharmacists (pharmacy technicians and pharmaceutical technical assistants) are permitted to advise on and sell medications in addition to pharmacists.’ Therefore, we believe that the sentence ‘Non-pharmacists (pharmaceutical technical assistants and pharmacy technicians) have a significantly higher likelihood of achieving the appropriate outcome than pharmacists.’ in the discussion section is now clearer. Point 2 must be also discussed in relation to their conclusion where they state “Furthermore, more questioning and involvement by non-pharmacists was identified as relevant factors influencing the appropriate outcome. There is a further need for research to identify why the advice provided is so often poor and particularly why the quality of advice provided by pharmacists is so inadequate”. Thank you for this comment. Please see our remarks to your point 3. We have deleted ‘but only under the supervision of pharmacists’ and have restructured the relevant sentence in the introduction section as follows: ‘In Germany, non-pharmacists (pharmacy technicians and pharmaceutical technical assistants) are permitted to advise on and sell medications in addition to pharmacists.’ Therefore, we believe that the sentences ‘Moreover, two or more questions and, surprisingly, the involvement of non-pharmacists were identified as relevant factors influencing the appropriate outcome.’ in the conclusions and ‘The findings of this study recommend further research to identify why the advice provided is so often poor and particularly why the quality of advice provided by pharmacists is so inadequate.’ in the study recommendations is easier to understand. In table 5, under Queue and Number of questions asked the percentages do not total to 100%. Therefore, the authors need to check their original data, or declare “Missing data” in the table to total the percentage to 100%. Thank you for this comment. We have checked all percentages reported in tables and made corrections if necessary."
}
]
},
{
"id": "58282",
"date": "11 Feb 2020",
"name": "Subish Palaian",
"expertise": [
"Reviewer Expertise Pharmacy Practice",
"Pharmacovigilance",
"Pharmacoepidemiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading the manuscript. It is a well conducted study with robust methodology and focusing on ‘community pharmacist referral’ which is an important aspect of community pharmacy practice. I thank the F1000Research for providing me the opportunity in reviewing this manuscript. The ‘simulated client method’ used in this research is suitable in evaluating the real-life practice happening at the community pharmacies.\n\nI have few concerns associated with the manuscript and are as below:\n\nAbstract:\n\nThe result section of the abstract mentions about ‘non-pharmacist’. It would be nice to elaborate more on those non-pharmacists somewhere in the Introduction section. Though it is mentioned in the Results, para 2, one would like to know whether non-pharmacists are allowed to dispense medications. This may be useful for international readers. Similarly, in the result part the statement ‘two or more questions as opposed to no questions were significantly associated with a referral to a doctor’ doesn’t appear to be novel. It is understood that someone who had not asked any question will be unable to refer. Authors might consider other observations to be mentioned at this section of the abstract.\n\nIntroduction:\n\nThe paragraph 6, authors need to clearly mention which ‘baseline’ and ‘follow-up’ study are they mentioning. What is the relation between the current study and those previous studies. Are they the same sample?\n\nI also found some similarity with few of the previous publications by the author using the same 'acute diarrhea' scenario. Authors need to verify the contents and avoid any similarities.\n\nMay be the Introduction section should elaborate more on the justification of this research. Why did all these studies used only ‘acute diarrhea’ as the scenario? This needs further elaboration.\n\nMethodology\nUnder scenario and assessment, it is not clear how authors categorized ‘Diarrhoea being present for five days as well as a high fever (40°C) are both considered to exceed the limits of self-medication’. I suppose even if there is presence of mucus and/or blood in the stool may be considered referral. I suggest more explanation on development of the scenario and content validation and why ‘Dysentery’ was excluded from the scenario.\n\nResults:\n\nIn this research, 45.2% of the dispensers were ‘non-pharmacists’. If that is the case then how can we correlate the research findings to evaluate the Community Pharmacy practice? The term ‘non-pharmacist’ needs more elaboration. Probably in the Introduction section, as I mentioned elsewhere.\n\nTable 4, response 8. Dispensing of a medication 175 (87.9%). What were these medications. Were there any antibiotics? What happened to the rest 22.1%? Why they did not dispense medications? These are some missing information and needs to be incorporated in the Results section.\n\n\nDiscussion:\nThe discussion can focus on reason for non-referral by the pharmacists. Are there skills related issues? Do the pharmacists possess adequate skills in assessing patients with diarrhea? These aspects may further enrich the discussion.\n\nLimitations:\nAuthors could explore any seasonal impact on the 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? Yes\n\nAre 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": "5449",
"date": "27 Apr 2020",
"name": "Bernhard Langer",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our manuscript and also for the very helpful and detailed comments, recommendations and questions. See our point-by-point response to the reviewer’s comments below. I enjoyed reading the manuscript. It is a well conducted study with robust methodology and focusing on ‘community pharmacist referral’ which is an important aspect of community pharmacy practice. I thank the F1000Research for providing me the opportunity in reviewing this manuscript. The ‘simulated client method’ used in this research is suitable in evaluating the real-life practice happening at the community pharmacies. I have few concerns associated with the manuscript and are as below: Abstract: The result section of the abstract mentions about ‘non-pharmacist’. It would be nice to elaborate more on those non-pharmacists somewhere in the Introduction section. Though it is mentioned in the Results, para 2, one would like to know whether non-pharmacists are allowed to dispense medications. This may be useful for international readers. Thank you for this comment. The following sentence has been added to the background section of the abstract: ‘In Germany, non-pharmacists (pharmacy technicians and pharmaceutical technical assistants) are permitted to advise on and sell medications in addition to pharmacists.’ Similarly, in the result part the statement ‘two or more questions as opposed to no questions were significantly associated with a referral to a doctor’ doesn’t appear to be novel. It is understood that someone who had not asked any question will be unable to refer. Authors might consider other observations to be mentioned at this section of the abstract. Thank you for this comment. We replaced the statement ‘two or more questions as opposed to no questions were significantly associated with a referral to a doctor’ with other observations. Introduction: The paragraph 6, authors need to clearly mention which ‘baseline’ and ‘follow-up’ study are they mentioning. What is the relation between the current study and those previous studies. Are they the same sample? Thank you for this comment. We expressed ourselves misleadingly.To clarify, the sentence has been restructured as follows: ‘Despite the two studies from 2014 and 2017 mentioned above, there remains a lack of information about the quality of advice for the indication acute diarrhoea in Germany. The reason is that the four scenarios investigated in these two studies were designed as ‘moderate’, in that referral to a medical consultation by the pharmacy staff was not mandatory in any of the scenarios.’ Also to clarify, the word ‘current’ has been added to this sentence: ‘The aim of this current study was, therefore, to determine on the basis of a considerably larger number of pharmacies whether the pharmacy staff referred patients to a medical consultation for a scenario in which consulting a doctor was mandatory (‘appropriate outcome’) and what the quality of questioning and – if a medication was dispensed – the quality of information provided were in this context.’ The following sentence was also added to the design subsection in the methods section: ‘It must once more be explicitly emphasized that this study shares no similarities with the previous studies conducted in 2014 and 2017 apart from the indication investigated (acute diarrhoea).’ I also found some similarity with few of the previous publications by the author using the same 'acute diarrhea' scenario. Authors need to verify the contents and avoid any similarities. Thank you for this comment. This study and the previous studies investigate the same indication (acute diarrhoea) but do not use the same scenario. As described in the introduction section, the scenarios used in previous studies from 2014 and 2017 were designed as ‘moderate’ in that in none of the scenarios was a referral for medical consultation by the pharmacy staff mandatory. In contrast, this study determines whether the pharmacy staff referred patients to a medical consultation in a scenario in which consulting a doctor was mandatory. Therefore, it is simulated – unlike the studies from 2014 and 2017 – that the grandmother has had diarrhoea for five days and fever (40°C) (see Table 1). To clarify, the following sentence has been added to the design subsection in the methods section: ‘It must once more be explicitly emphasized that this study shares no similarities with the previous studies conducted in 2014 and 2017 apart from the indication investigated (acute diarrhoea).’ May be the Introduction section should elaborate more on the justification of this research. Why did all these studies used only ‘acute diarrhea’ as the scenario? This needs further elaboration. Thank you for this comment. As mentioned in the introduction section, there are studies available from Germany regarding the quality of advice provided in pharmacies but none for this indication. To better clarify why three research studies for acute diarrhoea are necessary, the sentences have been restructured as follows: ‘Because of this lack of studies for acute diarrhoea, the quality of advice provided for acute diarrhoea in adults was analysed for Germany for the first time in 2014 and also identified clear deficiencies. Despite performance feedback used in the process to improve the quality of advice, a follow-up study in 2017 identical to that conducted in 2014 again showed poor quality of advice.’ And furthermore: ‘Despite the two studies from 2014 and 2017 mentioned above, there remains a lack of information about the quality of advice for the indication acute diarrhoea in Germany. The reason is that the four scenarios investigated in these two studies were designed as “moderate”, in that referral to a medical consultation by the pharmacy staff was not mandatory in any of the scenarios.’ Methodology Under scenario and assessment, it is not clear how authors categorized ‘Diarrhoea being present for five days as well as a high fever (40°C) are both considered to exceed the limits of self-medication’. I suppose even if there is presence of mucus and/or blood in the stool may be considered referral. I suggest more explanation on development of the scenario and content validation and why ‘Dysentery’ was excluded from the scenario. Thank you for this comment. More explanation on development of the scenario and content validation has been given. Additionally, it has been explained why ‘Dysentery’ was excluded from the scenario. Therefore, the relevant sentences have been restructured as follows: ‘The tool provided by the Federal Chamber of Pharmacies indicates 10 possible reasons (such as diarrhoea present > two to three days; fever > 39°C; blood or mucus in the stool; change from diarrhoea to constipation) each of which, if present, are considered to exceed the limits for self-medication and should result in the recommendation to consult a doctor. For this reason, a scenario was designed in which the grandmother’s diarrhoea had already been present for five days and she also had a fever of 40°C. Blood or mucus in the stool as another possible indication that the limits of self-medication had been exceeded was not considered in the scenario because it did not appear realistic for a grandmother to share this intimate information with her grandchildren. Therefore, if the pharmacy staff asked the SPs about the presence of blood in the grandmother’s stool, the SPs stated that it was not known whether there was blood in the stool.’ Results: In this research, 45.2% of the dispensers were ‘non-pharmacists’. If that is the case then how can we correlate the research findings to evaluate the Community Pharmacy practice? The term ‘non-pharmacist’ needs more elaboration. Probably in the Introduction section, as I mentioned elsewhere. Thank you for this comment. The following sentence has been added to the introduction section to better explain the term ‘non-pharmacist’: “In Germany, non-pharmacists (pharmacy technicians and pharmaceutical technical assistants) are permitted to advise on and sell medications in addition to pharmacists.’ Table 4, response 8. Dispensing of a medication 175 (87.9%). What were these medications. Were there any antibiotics? What happened to the rest 22.1%? Why they did not dispense medications? These are some missing information and needs to be incorporated in the Results section. Thank you for this comment. The relevant sentences have been restructured as follows: ‘In 87.9% (n=175) of all visits a medication was dispensed, whereas in 22.1% (n=24) of all visits no medication was dispensed. Regarding the visits in which a medication was dispensed, in 77.2% (n=135) of visits loperamide was dispensed and in 11.4% (n=20) probiotics were dispensed, whereas, for example, antibiotics that require a prescription in Germany were not dispensed at all.’ As described previously in the methods section, the guidelines expect that for such a condition that pharmacists are free to determine whether they also dispense a medication in addition to a mandatory referral to a doctor. It is therefore no wonder that a medication was not dispensed in all test purchases. Discussion: The discussion can focus on reason for non-referral by the pharmacists. Are there skills related issues? Do the pharmacists possess adequate skills in assessing patients with diarrhea? These aspects may further enrich the discussion. Thank you for this comment. To further enrich the discussion, the following sentence and an additional reference has been added: ‘In regards to a lack of knowledge, pharmacists should receive greater training during their studies in patient consultations using examples 44 , especially as to date the teaching of such ‘soft skills’ in German universities has been below average compared to other European countries 55 .’ Limitations: Authors could explore any seasonal impact on the finding. Thanks a lot for this comment. The following sentence and an additional reference to the limitations have been added: ‘Carrying out the visits in winter only may have had a seasonal impact on the findings because the pharmacy staff may possibly have been more aware of this indication due to the increased occurrence of acute diarrhoea in the winter months and therefore the results of our study may have been better than at other times of the year 74 .’"
}
]
}
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https://f1000research.com/articles/8-1841
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https://f1000research.com/articles/9-335/v1
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06 May 20
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{
"type": "Research Article",
"title": "Opportunistic screening for diabetes mellitus and hypertension in primary care settings in Karnataka, India: a few steps forward but still some way to go",
"authors": [
"Pracheth Raghuveer",
"Tanu Anand",
"Jaya Prasad Tripathy",
"Abhay Subhashrao Nirgude",
"Mahendra M. Reddy",
"Subhashree Nandy",
"Habeena Shaira",
"Poonam Ramesh Naik",
"Tanu Anand",
"Jaya Prasad Tripathy",
"Abhay Subhashrao Nirgude",
"Mahendra M. Reddy",
"Subhashree Nandy",
"Habeena Shaira",
"Poonam Ramesh Naik"
],
"abstract": "Background: Opportunistic screening for individuals aged ≥30 years at all levels of healthcare for early detection of diabetes mellitus (DM) and hypertension (HTN) is an integral strategy under the national program to control non-communicable diseases. There has been no systematic assessment of the screening process in primary care settings since its launch. The objective was to determine the number and proportion eligible for screening, number screened, diagnosed and treated for DM and HTN among persons aged ≥30 years in two selected primary health centres (PHCs) in Dakshina Kannada district, Karnataka, India during March-May 2019 and to explore the enablers and barriers in the implementation of screening from the perspective of the health care providers (HCPs) and beneficiaries . Methods: This was a sequential explanatory mixed-methods study with a quantitative (cohort design) and a descriptive qualitative component (in-depth interviews and focus group discussions) with HCPs and persons seeking care. Those that were not known DM/HTN and not screened for DM/HTN in one year were used to estimate persons eligible for screening. Results: Of 2697 persons, 512 (19%) were eligible for DM screening, 401 (78%) were screened; 88/401 (22%) were diagnosed and 67/88 (76%) were initiated on treatment. Of 2697, 337 (13%) were eligible for HTN screening, 327 (97%) were screened, 55 (17%) were diagnosed with HTN; of those diagnosed, 44/55 (80%) were initiated on treatment. The documentation changes helped in identifying the eligible population. Patient willingness to undergo screening and recognition of relevance of screening were screening enablers. Overworked staff, logistical and documentation issues, inadequate training were the barriers. Conclusion: Nearly 19% were eligible for DM screening and 13% were eligible for HTN screening. The yield of screening was high. We noted several enablers and barriers. The barriers require urgent attention to reduce the gaps in delivery and uptake of services.",
"keywords": [
"early detection",
"lifestyle diseases",
"opportunistic screening",
"operational research",
"SORT IT"
],
"content": "Introduction\n\nNon-communicable diseases (NCDs) kill 41 million people each year (71% of global deaths), disproportionately more (>75%) in the low-middle-income countries1. NCDs also account for a large share (75%) of deaths among those aged 30–69 years2. They are a threat to the Agenda for Sustainable Development 2030, which targets reduction of premature deaths from NCDs by one-third3.\n\nIndia mirrors the global picture, with NCDs claiming 63% of all deaths in 2016 alone2. India has nearly 72 million persons with diabetes mellitus (DM), which accounts for 49% of global burden and 207 million people with hypertension (HTN)4–7.\n\nEarly identification and prompt management through an emboldened health system is the key to reduce premature mortality and morbidity due to NCDs8. To achieve this, India launched the National Programme for Prevention and Control of Cancer, DM, Cardiovascular Diseases and Stroke (NPCDCS) in 20109. In Karnataka state, NPCDCS was introduced in a phased manner in various districts, starting from 2010–1110. In 2018, the programme was rolled out in Dakshina Kannada (DK) district, a coastal district in Karnataka. Opportunistic screening for persons aged ≥30 years at all public health facilities from sub-centres (SCs), primary health centres (PHCs) and above is an integral strategy for early detection of DM and HTN under the NPCDCS9.\n\nThere has been no systematic assessment of the screening process in programmatic settings, with previous studies conducted in project settings11–13. Furthermore, their focus was on the yield of screening. It is operationally important to know how many of the eligible population, could be screened, which to our knowledge has not been previously addressed11,13.\n\nTherefore, we conducted the present study among persons aged ≥30 years seeking health care from the outpatient department (OPD) of the selected PHCs in DK district of Karnataka from March to May, 2019 to determine i) the number and proportion eligible for screening of DM and HTN and ii) among those eligible, how many were screened, diagnosed and managed for the disease. Further, we qualitatively explored the enablers and barriers in the implementation of opportunistic screening from the perspective of the health care providers (HCPs) and persons availing the services.\n\n\nMethods\n\nThis was a sequential explanatory mixed-methods study with a quantitative component (cohort study) and a descriptive qualitative component14.\n\nGeneral setting. Karnataka is the eighth largest state of India and is inhabited by 61.1 million with a literacy rate of 75.4% and is divided into 30 administrative districts15.\n\nDK, a coastal district of Karnataka, has a population of ~2.1 million and a literacy rate of 85.3%. It is divided into nine administrative divisions called Talukas16. The prevalence of DM and HTN in DK are 16% and 17% respectively, higher than the national figures17,18.\n\nSpecific setting. Mangaluru is a predominantly urban Taluka of DK district with a population of ~1 million and a literacy rate of 91%19. It has 22 PHCs and 12 urban primary health centres (UPHCs) which deliver primary health care to the population. We selected one UPHC located in Bunder, which caters to a population of 6,749 and one PHC located in Amblamogaru, a rural area with a population of 16,920. Yenepoya Medical College, where the Principal Investigator (PI) works, supports these centres by posting medical interns, as per a Memorandum of Understanding with the District Health and Family Welfare Office, DK.\n\nOpportunistic screening process for DM and HTN at the PHC level\n\nThe PHCs run a general OPD where the basic demographics, diagnosis and treatment details are recorded in the OPD register. Under the NPCDCS, opportunistic screening is being conducted by the staff nurse under the supervision of the Medical Officer (MO) and details are recorded in a separate register (NCD register).The laboratory technician plays a supporting role in opportunistic screening for DM by carrying out tests like random blood glucose (using a glucometer) and fasting blood sugar (FBS), and maintains records of the tests conducted. An additional NCD related activity being carried out in these PHCs include population-based screening (PBS). PBS is carried out by accredited social health activists (ASHAs) through home visits in their service areas and by auxiliary nurse midwives (ANMs) at the SC level.\n\nMonthly reports of all NCD-related activities at the PHC level are collated in a reporting format which captures details like cumulative number of persons screened, diagnosed, treated and on follow-up care for DM, HTN and other NCDs. This report is submitted to the district NCD cell, which is responsible for effective implementation and supervision at the district level. The NCD cell is managed by the District Programme Coordinator of NPCDCS, who works under the overall supervision of the District Surveillance Officer (DSO).\n\nFor the quantitative phase all persons aged ≥30 years availing primary health care from the two selected PHCs from March to May 2019 were included. We excluded persons aged <30 years who sought primary health care from the two selected PHCs.\n\nFor the qualitative phase, HCPs working in the two selected PHCs, who were involved in screening for DM and HTN like staff nurses (n=4), laboratory technicians (n=2) and MO of the PHCs (n=3) were included. HCPs who were not involved in the screening process for DM and HTN at the two PHCs were excluded. The District Programme Coordinator, NPCDCS (n=1) was also interviewed. Persons who underwent DM and HN screening in the two PHCs from March to May 2019 (n=37) also constituted the study population.\n\nPhase 1: Quantitative data collection\n\nSetting up of a system for better documentation of opportunistic screening for DM and HTN at the selected PHCs\n\nExperiences from the field show that the existing recording system to document opportunistic screening carried has certain limitations, particularly with respect to determining the population eligible for screening among persons aged ≥30 years. It is not well documented whether a person has undergone screening previously or is already diagnosed as DM or HTN.\n\nThus, we set up a system to improve the existing documentation for opportunistic screening of DM and HTN.\n\nAfter obtaining necessary permissions and building initial rapport with the HCPs, we conducted a stakeholder meeting at the PHCs. The limitations of the current recording system were discussed and additional variables were included in both the OPD and NCD registers. The variables include: a) whether the person has DM/HTN, b) whether screened for DM/HTN in the last one year. If the response to both a) and b) were “no”, the person was considered to be eligible for screening. The was done to estimate the number of persons eligible for screening and to assess the feasibility of this strategy in such settings. Further, we made amendments in the NCD register to collect certain essential information.\n\nThe staff nurses and laboratory technicians were trained to enter the required information in dichotomous responses (Yes/No). This enabled us to assess the eligibility for screening.\n\nData collection\n\nThe screening process was implemented by the HCPs from March to May 2019 at the two PHCs. To mitigate bias, none of the members of the study team were in contact with the HCPs of the two PHCs during the above-mentioned period of implementation. Thereafter, we collected details from the OPD and the NCD registers for the duration, March to May 2019 in a structured data collection proform a (available as Extended data20) which had two parts. Data for the first part were extracted from the OPD register and data for the second part data came from the NCD register. The first part collected demographic details and eligibility criteria for screening. The second part collected information on whether persons were screened, diagnosed or managed for DM and HTN. Epidemiological diagnosis for DM, HTN and eligibility for screening are given in Table 1.\n\nPhase 2: Qualitative data collection. Systematic qualitative enquiry was carried out through key informant interviews (KIIs) among HCPs and focus group discussions (FGDs) among persons aged ≥30 years, who underwent screening for DM and HTN.\n\nThe PI has a master’s degree in Community Medicine/Public Health and is trained in qualitative research methods. The investigators were not a part of the programme implementation team.\n\nThe PI conducted the KIIs among HCPs at their workplace in Kannada (vernacular language), or English as applicable, until information saturation was attained. Participants were explained the purpose and their expected role prior to the interview about. Interview guides consisting of broad open-ended questions and probes were prepared for different cadres. Each KII lasted for around 30 minutes. Interview and FGD guides are available as Extended data33.\n\nThe PI also conducted FGDs among persons aged ≥30 years, who underwent screening at the PHC. A total of 6–8 participants were included in each FGD. FGDs. Each FGD lasted for about 45 minutes and were held in Kannada language separately for men and women.\n\nOnly the participants, the PI and the note-maker were present during the KIIs and FGDs. Audio recording and verbatim notes were taken. In case the participants did not consent for audio recording prior to the discussion, notes were taken. After the KII/FGD was over, the summary was read back to the participants to ensure validation. A total of two repeat interviews were conducted among a staff nurse and laboratory technician working in one of the two PHCs. A repeat FGD was conducted among men aged ≥30 years who underwent screening for DM and HTN in one of the PHCs.\n\nQuantitative data. Quantitative data were double-entered and validated using Epi Data version 3.1 for entry. The data was analysed using Epi Data version 2.2.2.183 (Epi Data Association, Odense, Denmark) and STATA (v12.1) software.\n\nContinuous data were summarized using mean and standard deviation (SD). Categorical data were summarized as proportions. Key indicators like proportion of eligible population screened, diagnosed and managed for DM and HTN are presented in a flow diagram (Figure 1 and Figure 2). To assess the factors associated with ‘not screened for DM and HTN’, we used Poisson regression. Adjusted relative risks (aRRs) with 95% confidence intervals (95% CIs) were calculated to eliminate the confounders. A p value of <0.05 was considered as the criterion of statistical significance.\n\nQualitative data. The audio-recorded interviews and FGDs were transcribed by the PI (PR) in Kannada within 48 hours. Thematic analysis by manual coding was carried out by three researchers (PR, ASN and SN) independently to generate various categories or themes under the broad topics: HCP-related and patient-related enablers and barriers. Any discrepancy in coding was resolved through discussion and referral back to the audio files if necessary. If the discrepancy was still not resolved, a third investigator (PRN) reviewed the transcripts and codes. The transcripts and analysis were reviewed by other investigators (TA, JT) to reduce subjectivity in analysis and increase interpretive credibility. The codes were then organised into categories and common themes and presented in flow diagrams (Figure 3 and Figure 4). A mix of inductive and deductive coding was done. Verbatim quotes are also presented (translated into English) within double quotations23,24. To ensure confidentiality, we have deliberately not mentioned the designation of HCPs in the quotes. The findings have been reported by using ‘Consolidated Criteria for Reporting Qualitative Research’ (COREQ) guidelines25.\n\nEthics approval was received from Yenepoya Ethics Committee-1,Yenepoya (Deemed to be University), Mangaluru (2019/085)and the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease, Paris, France (126/18). Written informed consent was obtained from the study participants interviewed. Permission to carry out the study was obtained from the District Health and Family Welfare Officer, DK district.\n\n\nResults\n\nOf the total 4120 persons seeking health care, 2697 fulfilled the eligibility for the study and were included in the analysis.\n\nOf the 2697, 812 (30.2%), were aged 30–39 years with a mean age of 47.7 years (SD:12.3 years). More than half of the respondents were males (1525, 56.5%); nearly half were from UPHC (1350, 50.0%) (Table 2).\n\nOf the 2697, 1631 (60.5%) were reported to have been screened for DM in the last year. A total of 512 (19%) were eligible for DM screening, among which 401 (78.3%) were screened for DM of whom, 88 (21.9%) were diagnosed as DM. Of the 88 diagnosed as DM, 67 (76.1%) were initiated on treatment (Figure 1).\n\nOf the 2697, majority (1623, 60.2%) had already been screened for HTN in the last year. A total of 337 (12.5%) were eligible for HTN screening. Of the 337, 327 (97%) were screened for HTN, of whom, 55 (16.8%) were diagnosed with HTN. Of the 55 diagnosed with HTN, 44 (80%) were initiated on treatment (Figure 2).\n\nIn the adjusted analysis, female gender (aRR: 1.3, 95% CI: 1.0-1.8, p-value 0.04) was independently associated with ‘not being screened for DM’ (Table 3). Male gender (RR: 2.4, 95% CI: 0.5-10.9, p-value 0.3) was not significantly associated with ‘not being screened for HTN’ (Table 4). De-identified participant information is available as Underlying data26.\n\naRR, adjusted relative risk; CI, confidence interval.\n\naRR, adjusted relative risk; CI, confidence interval\n\nQualitative. Opportunistic screening was acknowledged by HCPs and persons screened for DM and HTN as a useful strategy for early detection. We have summarized the potential enablers and barriers for implementation of opportunistic screening for DM and HTN under two broad organizing themes, HCP-related (health care staff of the PHCs and District Programme Coordinator, NPCDCS) and patient-related (persons screened for DM and HTN from March to May 2019 in the two PHCs)(Figure 3 and Figure 4).\n\nKnowledge of screening. The HCPs demonstrated satisfactory knowledge of the process and relevance of screening and acknowledged its role in early detection.\n\n“Now they (adult population) are coming early. Early screening is better because the disease onset is early and if undetected, could lead to complications.” (HCP, 34 years, female)\n\nWillingness to deliver services. HCPs expressed willingness to implement opportunistic screening in their settings, despite facing challenges like staff shortages.\n\n“We have trained all staff including attendants on Glucometer usage. Therefore, the screening is going on smoothly despite staff shortage.” (HCP, 55 years, male)\n\nFurther, the HCPs believed that it is imperative to screen more often. One HCP stated that it is prudent to screen persons for DM and HTN at least once in six months.\n\n“We need to change to once in six months screening as once a year is inadequate. Over a period of time, the risk may increase.” (HCP, 44 years, female)\n\nStrength of PBS. PBS is being implemented in the community by the ANMs and ASHAs, through which many persons are being screened.\n\n“I strongly feel that PBS is the strength of NPCDCS at least in our district.” (HCP, 38 years, female).\n\nChanges for better documentation. Many HCPs welcomed the changes made in the OPD and NCD registers and were of the view that this improved the documentation of opportunistic screening.\n\n“It is absolutely fine. We get to know the eligible patients who really require screening.” (HCP, 29 years, female)\n\nMost of them did not experience any problems in recording the details mentioned in the registers.\n\n“No issues with the new documentation system. I could maintain both the registers properly.” (HCP, 29 years, female)\n\nAwareness of screening. Persons who underwent screening understood its role in early detection, facilitating prompt treatment and preventing complications.\n\n“The earlier we get diagnosed, the sooner we are treated. We must get screened before [the blood sugars and pressure]become high.” (patient, 52 years, male)\n\nReadiness to undergo screening. Most of the participants, expressed readiness to undergo screening, despite their values falling within normal limits. Many were willing to come for confirmatory tests if required. One participant indicated that she has followed dietary advice as a prevention for DM and HTN.\n\n“I get tested frequently even though I do not have disease. I have controlled my food habits just to be careful.” (patient, 60 years, female)\n\nSatisfaction with health care. Many persons were satisfied with the screening services. Two persons mentioned that they had not faced problems while undergoing screening at the PHC and were happy with the attitude of the HCPs.\n\n“No problems here. All staff are good.” (patient, 61 years, male)\n\n“I haven’t faced problems. They check blood pressure and sugar properly.” (patient, 44 years, female)\n\nIn spite of acceptance of this initiative by both HCPs and persons undergoing screening, several implementation barriers were noted.\n\nStaff challenges. Many HCPs acknowledged increase in workload and inadequate staff as significant implementation challenges. Vacant posts, high proportion of persons with DM and HTN and the pressure of other health programmes were perceived to be the challenges.\n\n“Too many programmes and many patients. We are expected to check weight and height as well. Where do we get the time for all that?” (HCP, 29 years, female)\n\n“Inadequate staff is a huge concern. Many PHCs do not have adequate staff nurses and laboratory technicians.” (HCP, 38 years, female)\n\nLack of adequate human resources is the main issue, as stated by one HCP. To address this, task shifting is being practised with multiple personnel involved in documentation and screening.\n\n“We do not have enough staff. The data entry operator was on maternity leave for a long time. Data entry is now done by staff nurse as the data entry operator is overburdened. When she is not there, the laboratory technician or pharmacist contribute.” (HCP, 44 years, female)\n\nLogistical issues. Some HCPs expressed concerns over shortage of strips (Glucometer) and medicines once in a while. One HCP felt that facilities to transport patients/blood samples to a higher centre should be available, in case of non-availability of diagnostics at PHC.\n\n“Another issue is the shortage of NCD drugs. I end up prescribing for fifteen days instead of a month, which is not ideal.” (HCP, 44 years, female)\n\nTimely allocation and release of budget is another major barrier. Administrative delay was a contributing factor to the delay in release of funds and supply of equipment.\n\n“If we don’t have money, the implementation becomes difficult. Last year, the budget was approved on time but the money came only in December.” (HCP, 38 years, female)\n\n“Things do not come on time and government procedures are lengthy.” (HCP, 55 years, male)\n\nDocumentation and reporting challenges. High patient load and lengthy reporting format were the documentation-related challenges, as stated by a HCP. Another HCP suggested recruitment of dedicated staff for documentation and reporting.\n\n“The reporting format is complicated and consumes a lot of time.” (HCP, 35 years, female)\n\n“The registers given by the programme are lengthy. It includes not just diabetes and hypertension but other NCDs like breast, cervical and oral cancers. All our staff are busy with other health programmes.” (HCP, 32 years, female)\n\nDelay in submission of reports was another issue that was highlighted by a participant.\n\n“We don’t get the reports on time. Reports have to reach by the 5th of every month. But there is always a delay.” (HCP, 38 years, female)\n\nInadequate training. Training conducted at the District NCD Cell focused more on treatment and indent of logistics while issues like screening were neglected. Need to organize comprehensive training programmes on screening and documentation was noticed.\n\n“I was not trained on how to conduct screening. The training focused on treatment and not on documentation and screening.” (HCP, 32 years, female)\n\nFrequent screening demand. Few HCPs opined that patients demand tests frequently. Thus, it is difficult to restrict screening to once a year.\n\n“If we make it (screening) once a year, many go and complain. If we do not agree to the patients’ demands, they complain to the corporator (elected public representative). I wonder if this would work.” (HCP, 29 years, female)\n\nLow uptake among women. HCPs stated that few women expressed difficulties in undergoing FBS the next morning.\n\n“Women give excuses and don’t turn up the next morning for FBS, despite we counselling them.” (HCP, 35 years, female)\n\n“Women mention they have household work and refuse tests. I also feel that they are more anxious.” (HCP, 55 years, male)\n\nReluctance for FBS. Many persons with high random blood glucose did not undergo FBS. One person felt that since glucometer testing is done, FBS on the next morning may be redundant. Another person stated preoccupation with work and late opening time of the centre as reasons for refusal.\n\n“I get it (sugars) checked in Glucometer, so what is the need of fasting sugars?” (patient, 50 years, female)\n\n“PHC opens only at 9 am. I have to report for my work at that time.” (patient, 52 years, male)\n\nWaiting time and inconvenience. Few persons expressed their unhappiness about the waiting period for reports and consulting the MO.\n\n“By the time I finish my household work and come to the centre, the senior doctor would have left. Then, I have to wait for the next doctor in the evening (evening clinic).” (patient, 39 years, female)\n\nTwo of the participants were apprehensive about the health care staff drawing the blood repeatedly, which caused them inconvenience.\n\n“Sometimes, they check our sugars despite getting it done recently. Why unnecessary take blood and subject us to more stress? ” (patient, 50 years, male)\n\n“They prick thrice to collect blood. I fast overnight. It is difficult to withstand.” (patient, 46 years, female)\n\nChallenges faced by women. A least two women mentioned that it is difficult for them to make repeat visits to the PHC for testing, especially in the morning.\n\n“Here, it opens very late, at around 9 am. It is difficult for me, as I need to drop my children to school.” (patient, 50 years, female)\n\n“I have to go for work at a factory after the household work. So, how will I be able to come in the morning?” (patient, 37 years, female)\n\nShortage of medicines. Persons diagnosed with DM/HTN and started on treatment expressed concern regarding shortage of medicines for DM and HTN in the PHC.\n\n“They prescribe medicines for just 10–15 days and ask us to come back. It is a disturbance for us.” (patient, 47 years, male)\n\n“They say there are no medicines here. They do not give for more than a week.” (patient, 61 years, male)\n\nDe-identified transcripts from interviews and FGDs are available as Underlying data26.\n\n\nDiscussion\n\nTo our knowledge, this is one of the first mixed methods studies from India assessing the implementation of opportunistic screening for DM and HTN under NPCDCS in primary care settings. We made certain amendments in the OPD register to capture the population eligible for screening and in the NCD register to determine the number screened, diagnosed and treated for DM and HTN. We found that 19% were eligible for DM screening, of which 78% underwent screening and 13% were eligible for HTN screening, among whom 97% were screened. Willingness for screening both on the part of HCPs and persons seeking health care was a key facilitator. Several barriers like staff, logistics, documentation and waiting time were noted. The key findings are discussed below.\n\nFirst, we found that a substantially low proportion were eligible for opportunistic screening (19.0% for DM and 13.0% for HTN). More than half were screened for DM and HTN in the last year. This is probably due to the PBS conducted in the rural community, an ongoing activity under carried out by ANMs/ASHAs who approach persons aged ≥30 years in the community through home visits or outreach camps. Community-based assessment checklists (CBAC) are filled out and those with high risk are referred to the SC for screening. If found positive, they are referred to the PHC for further investigations and treatment27. Further, in urban areas of Mangaluru, special outreach camps with a focus on screening for DM and HTN are carried out once a month, which could have contributed to our finding of low proportion of eligible population.\n\nSecond, nearly 22% of the population screened were diagnosed with DM and 19% were diagnosed with HTN, which is much higher than the National Family Health Survey-4 (NFHS-4) data for DK district, in which ≈7.0% had high blood sugar and ≈12.0% had hypertension28. This could be ascribed to the fact that our study was a facility-based assessment while NFHS-4 was a community-based survey. Similarly, a community-based survey in coastal Karnataka reported the prevalence of DM to be 16%, lower than the yield in our study. (19) A study conducted in a semi-urban population of Mangaluru reported a prevalence of 41% hypertension, which was much higher than our finding29. Despite these variations, the high burden of DM and HTN is a matter of concern which requires both population and individual level interventions.\n\nThird, women were more likely ‘not to be screened’ for DM when compared to men. This finding of our study could be attributed to the fact that women may be preoccupied with household work. This was substantiated in the qualitative component, where women listed reasons like domestic work and looking after children for not undergoing FBS. It could also be speculated that women are more likely to prioritize their family and may tend to neglect their own health. A qualitative study which assessed the barriers for screening of DM among Iranian women found that many women perceived screening for DM as difficult and also expressed reluctance to undergo blood sugar testing30.\n\nFourth, we found that both the PHCs were staffed by HCPs who displayed a positive attitude towards delivery of NCD screening services. We also found that many persons seeking health care expressed readiness to undergo screening. Willingness is an important predictor for the success of screening for DM and HTN, as reported by previous studies31,32. The key reason for this finding could be the good rapport that the HCPs shared with the community.\n\nFifth, most of the HCPs were satisfied with the amendments made in both the OPD and NCD registers and believed that this made their job easier in terms of determining the eligible population. One drawback of the registers prescribed by the programme is that the eligible population could not be identified. The HCPs felt that the NCD registers prescribed by the programme include too many variables. We have tried to address this through modifications in the recording registers.\n\nSixth, few HCPs recommended half-yearly screening for persons without DM and HTN. This would lead to unnecessary screening and wasted resources There is a need to sensitize HCPs on restricting to once a year screening for judicious use of resources. The NPCDCS training manuals also advocate screening once a year for DM and HTN among the general population21,27. This needs to be emphasized in future training programmes conducted under the NPCDCS.\n\nSeventh, staff challenges, logistical issues and documentation issues were the major barriers, as perceived by HCPs. The health care staff seem to be overburdened with many programmes. This is likely to affect their productivity and in turn hamper the implementation of opportunistic screening. Further, timely submission of reports to the district NCD cell becomes difficult.\n\nEighth, despite being aware of the relevance, many eligible persons failed to get themselves screened. Moreover, many who screened positive for DM did not undergo FBS. This was mainly due to preoccupation with work in the morning hours. Fear and uncertainty surrounding test results may have further contributed to this attrition. It is imperative to sensitize persons seeking care about the importance of FBS as a diagnostic test.\n\nIncreased waiting time was another challenge. It was also noted that laboratory technicians get deputed to other PHCs on certain days to address the issue of staff shortage. This may affect timely reporting of tests like FBS, which in turn results in a missed opportunity to initiate prompt treatment of DM.\n\nThis is the first study providing information on persons eligible for opportunistic screening in a primary care setting. Our study was conducted under programmatic conditions and the findings reflect the ground realities. We have used a sequential mixed-methods design, which helped in a comprehensive assessment of the enablers and barriers for implementation to guide further refinement of the programme. This will guide the programme managers to take corrective measures.\n\nMost of the studies on this topic are focussed on population-based screening approaches and do not highlight facility-based implementation challenges. Since the investigators were not a part of the programme implementation team, this ensured objectivity in analysis and interpretation. Further, we included all persons aged ≥30 years seeking health care from the two PHCs, thereby ensuring internal validity. We adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) and COREQ guidelines for reporting quantitative and qualitative components, respectively25,33.\n\nThe findings of our study need to be interpreted cautiously as it was conducted in two PHCs.The findings cannot be extrapolated to other settings or geographical areas. There were some gaps in accurately recording the information about having underwent opportunistic screening in the last one year. There could be an element of recall bias as this was a self-reported variable.\n\nFirst, urgent attention should be given to address staff challenges which includes filling of vacant posts and hands-on training for documentation. Second, we need to capitalize on the health seeking behaviour of persons seeking health care by timely delivery of services. Third, we need to nurture positive attitudes in HCPs by supportive supervision, training, regular supply of medicines and provision of incentives. Fourth, some eligible beneficiaries werenot screened. This needs to be addressed by digital solutions like line listing of the eligible population. Fifth, the modifications that we made in the registers helped in identifying the eligible population. However, this needs cautious interpretation and may require further studies before being implemented across all PHCs.\n\n\nConclusion\n\nOur study found a low proportion eligible for DM and HTN screening. Among those screened, a high number had DM and HTN. We made modifications in the documentation of screening which were well-received by the HCPs. We observed several enablers and barriers to implementation of opportunistic screening. The NPCDCS must address the barriers if it has to strengthen opportunistic screening in primary care settings.\n\n\nData availability\n\nFigshare: Opportunistic screening for diabetes mellitus and hypertension in primary care settings of Karnataka, India: few steps forward but still some way to go- Raw Data.\n\nhttp://doi.org/10.6084/m9.figshare.1205295026.\n\nThis project contains the following underlying data:\n\nOpportunistic screening DM HTN_OPD spreadsheet. (Data of 2697 persons who availed primary health care extracted from the OPD registers of two PHCs)\n\nOpportunistic screening DM HTN_NCD spreadsheet. (Data of 529 persons who were eligible for diabetes mellitus/hypertension screening extracted from the NCD registers of two PHCs)\n\nOpportunistic screening DM HTN_Data Documentation Sheet. (Data Documentation sheet used to code the variables)\n\nKey Informant Interview-Programme Coordinator. (Transcript of the Key Informant Interview conducted on the Programme Coordinator of the district)\n\nKey Informant Interview-Medical Officer 1. (Transcript of the Key Informant Interview conducted on Medical Officer of one of the two selected PHCs)\n\nKey Informant Interview-Medical Officer 2. (Transcript of the Key Informant Interview conducted on Medical Officer of one of the two selected PHCs)\n\nKey Informant Interview-Medical Officer 3. (Transcript of the Key Informant Interview conducted on Medical Officer involved in the non-communicable disease programme)\n\nKey Informant Interview- Staff Nurse 1 (Transcript of the Key Informant Interview conducted on Staff Nurse of one of the two selected PHCs)\n\nKey Informant Interview- Staff Nurse 2 (Transcript of the Key Informant Interview conducted on Staff Nurse of one of the two selected PHCs)\n\nKey Informant Interview- Staff Nurse 3 (Transcript of the Key Informant Interview conducted on Staff Nurse of one of the two selected PHCs)\n\nKey Informant Interview- Staff Nurse 4 (Transcript of the Key Informant Interview conducted on Staff Nurse of one of the two selected PHCs)\n\nKey Informant Interview- Laboratory Technician 1 (Transcript of the Key Informant Interview conducted on Laboratory Technician of one of the two selected PHCs)\n\nKey Informant Interview- Laboratory Technician 2 (Transcript of the Key Informant Interview conducted on Laboratory Technician of one of the two selected PHCs)\n\nFocused Group Interview-Patients 1 (Transcript of the Focused Group Discussion conducted on persons screened for diabetes mellitus or hypertension in one of the two selected PHCs)\n\nFocused Group Interview-Patients 2 (Transcript of the Focused Group Discussion conducted on persons screened for diabetes mellitus or hypertension in one of the two selected PHCs)\n\nFocused Group Interview-Patients 3 (Transcript of the Focused Group Discussion conducted on persons screened for diabetes mellitus or hypertension in one of the two selected PHCs)\n\nFocused Group Interview-Patients 4 (Transcript of the Focused Group Discussion conducted on persons screened for diabetes mellitus or hypertension in one of the two selected PHCs)\n\nFocused Group Interview-Patients 5 (Transcript of the Focused Group Discussion conducted on persons screened for diabetes mellitus or hypertension in one of the two selected PHCs)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Opportunistic screening for diabetes mellitus and hypertension in primary care settings of Karnataka, India : few steps forward but still some way to go- Extended Data\n\nhttp://doi.org/10.6084/m9.figshare.1205305520.\n\nThis project contains the following underlying data:\n\nStudy Proforma (Data collection proforma used in the study)\n\nKey Informant Interview Checklist (Checklist used for the Key Informant Interviews)\n\nFocused Group Discussion Guide (Guide used for the Focused Group Discussions)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership led by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR). The model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF/Doctors Without Borders). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: The Union South-East Asia Office, New Delhi, India; the Centre for Operational Research, The Union, Paris, France; Medécins sans Frontières (MSF/Doctors Without Borders), India; Department of Preventive and Social Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India; Department of Community Medicine, All India Institute of Medical Sciences, Nagpur, India; Department of Community Medicine, ESIC Medical College and PGIMSR, Bengaluru, India; Department of Community Medicine, Sri Manakula Vinayagar Medical College and Hospital, Puducherry, India; Karuna Trust, Bangalore, India; Public Health Foundation of India, Gurgaon, India; The INCLEN Trust International, New Delhi, India; Indian Council of Medical Research (ICMR), Department of Health Research, Ministry of Health and Family Welfare, New Delhi, India; Department of Community Medicine, Sri Devraj Urs Medical College, Kolar, India; and Department of Community Medicine, Yenepoya Medical College, Mangalore, India.\n\nWe would like to thank the study participants who kindly agreed to share their insights about the enablers and barriers.We would like to thank the health care providers of the two primary health centres for their active contribution and support. We are also grateful to have received the encouragement and support by Dr. Ramakrishna Rao (District Health and Family Welfare Officer, Dakshina Kannada district) and Dr. MS Moosabba (Principal/Dean, Yenepoya Medical College). We acknowledge the contribution of Dr. Shwetha Nemgoudar, Dr. Sharmila, Dr. Nashath Farhana, Dr. Noorshaba Anwar, Dr. Noushima, Dr.Shivakumar, Dr. Rakesh V, Dr. Rajesh DN, Dr. Rakshan Shetty, Dr. Sachin, Dr. Naseem Ashraf, Dr. Puneet Saini and Dr. Arun (medical interns, Yenepoya Medical College) for their help in data collection and entry.\n\nStudy protocol: PR, TA, JPT, ASN, MMR, SN, HS, PRN\n\nData collection: PR, SN, HS\n\nData analysis and interpretation: PR, JPT, ASN, MMR\n\nDrafting a paper: PR, JPT, ASN\n\nCritically reviewing the paper and approving the final version: PR, TA, JPT, ASN, PRN\n\nOpinions, discussions, views and recommendations expressed in this article are solely those of the authors and do not necessarily represent that of the organizations they are affiliated with.\n\n\nReferences\n\nWorld Health Organization: Global Action Plan 2013-2020. World Health Organization, Geneva; 2013; 55. Reference Source\n\nWorld Heallth Organization: Noncommunicable Diseases Country Profiles 2018. Geneva; 2018; 223. Reference Source\n\nUnited Nations: Transforming Our World: The 2030 Agenda for Sustainable Development. New York; 2015. Reference Source\n\nInternational Diabetes Federation: IDF SEA Region. 2018; [cited 2018 Nov 16]. Reference Source\n\nIndia State-Level Disease Burden Initiative Collaborators: Nations within a nation: variations in epidemiological transition across the states of India, 1990-2016 in the Global Burden of Disease Study. Lancet. 2017; 390(10111): 2437–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiegel KR, Patel SA, Ali MK: Non-communicable diseases in South Asia: contemporary perspectives. Br Med Bull. 2014; 111(1): 31–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta R, Gaur K, S Ram CV: Emerging trends in hypertension epidemiology in India. J Hum Hypertens. 2019; 33(8): 575–587. PubMed Abstract | Publisher Full Text\n\nCao B, Bray F, Ilbawi A, et al.: Impact on life expectancy of a one-third reduction in premature mortality from NCDs by 2030: a global analysis of the Sustainable Development Goal health target. Lancet Glob Health. 2018; 6(12): e1288–96.\n\nMinsitry of Health and Family Welfare Government of India: NPCDCS Operational Revised Guidelines 2013-2017. 2017; 130. Reference Source\n\nGovernment of Karnataka: National Programme for Prevention and Control of Cancer, Diabetes, Cardiovascular Diseases and Stroke (NPCDCS). 2016; [cited 2019 Aug 12]. Reference Source\n\nAmarchand R, Krishnan A, Saraf DS, et al.: Lessons for addressing noncommunicable diseases within a primary health-care system from the Ballabgarh project, India. WHO South East Asia J Public Health. 2015; 4(2): 130–138. PubMed Abstract | Publisher Full Text\n\nAnand T, Kishore J, Isaakidis P, et al.: Integrating screening for non-communicable diseases and their risk factors in routine tuberculosis care in Delhi, India: A mixed-methods study. PLoS One. 2018; 13(8): e0202256. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajumdar A, Chinnakali P, Vinayagamoorthy V, et al.: Opportunistic Screening for Hypertension and Selected Cardiovascular Risk Factors among Adults Attending a Primary Health Center in Puducherry, India. Int J Prev Med. 2014; 5(12): 1616–20. PubMed Abstract | Free Full Text\n\nMorse JM: Approaches to qualitative-quantitative methodological triangulation. Nurs Res. 1991; 40(2): 120–3. PubMed Abstract | Publisher Full Text\n\nGovernment of India Ministry of Home Affairs Office of Registrar General and Census Commissioner: Size, Growth Rate and Distribution of Population. 2011. Reference Source\n\nGovernment of India Ministry of Home Affairs Office of Registrar General and Census Commissioner: Dakshina Kannada District Population Census-Details 2011 Data. 2018. Reference Source\n\nRao CR, Kamath VG, Shetty A, et al.: A study on the prevalence of type 2 diabetes in coastal Karnataka. Int J Diabetes Dev Ctries. 2010; 30(2): 80–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParthaje PM, Unnikrishnan B, Thankappan KR, et al.: Prevalence and Correlates of Prehypertension Among Adults in Urban South India. Asia Pac J Public Health. 2016; 28: 93S–101S. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGovernment of India Ministry of Home Affairs Office of Registrar General and Census Commissioner: Mangalore Taluka Population, Caste, Religion Data - Dakshina Kannada district, Karnataka. 2018; Reference Source\n\nCreswell J, Plano Clark V: Designing and Conducting Mixed Methods Research. London (United Kingdom): Sage Publications Ltd; 2007; 142–5. Reference Source\n\nKvale S: Doing interviews. London (United Kingdom): SAGE Publications; 2007; 142. Reference Source\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care. 2007; 19(6): 349–57. PubMed Abstract | Publisher Full Text\n\nNational Health Mission: Module for Multi-Purpose Workers (MPW)-Female/Male on Prevention, Screening and Control of Common Non-Communicable Diseases. New Delhi; 2018. Reference Source\n\nMinistry of Health and Family Welfare: National Family Health Survey - 4 District Fact Sheet Stana Madhya Pradesh. 2015. Reference Source\n\nAdhikari P, Pemminati S, Pathak R, et al.: Prevalence of Hypertension in Boloor Diabetes Study (BDS-II) and its Risk Factors. J Clin Diagn Res. 2015; 9(11): IC01–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRafii F, Rahimparvar SFV, Mehrdad N, et al.: Barriers to postpartum screening for type 2 diabetes: a qualitative study of women with previous gestational diabetes. Pan Afr Med J. 2017; 26: 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNijhof N, ter Hoeven CL, de Jong MD: Determinants of the use of a diabetes risk-screening test. J Community Health. 2008; 33(5): 313–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNelissen HE, Okwor TJ, Khalidson O, et al.: Low uptake of hypertension care after community hypertension screening events in Lagos, Nigeria. Glob Health Action. 2018; 11(1): 1548006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Health and Family Welfare Government of India: National Programme for Prevention and Control of Cancer, Diabetes, Cardiovascular Diseases and Stroke Training Module for Medical Officers for Prevention, Control and Population Level Screening of Hypertension, Diabetes and Common Cancer (Oral, Breast & Cervical). New Delhi; 2017. Reference Source\n\nVandenbroucke JP, von Elm E, Altman DG, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration. Int J Surg. 2014; 12(12): 1500–24. PubMed Abstract | Publisher Full Text\n\nJames PA, Oparil S, Carter BL, et al.: 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014; 311(5): 507–20. PubMed Abstract | Publisher Full Text\n\nRaghuveer P, Anand T, Tripathy JP, et al.: Opportunistic screening for diabetes mellitus and hypertension in primary care settings of Karnataka, India: few steps forward but still some way to go- Raw Data. figshare. 2020; Dataset. http://www.doi.org/10.6084/m9.figshare.12052950.v3\n\nRaghuveer P, Anand T, Tripathy JP, et al.: Opportunistic screening for diabetes mellitus and hypertension in primary care settings of Karnataka, India: few steps forward but still some way to go- Extended Data. figshare. 2020; Dataset. http://www.doi.org/10.6084/m9.figshare.12053055.v1"
}
|
[
{
"id": "63174",
"date": "14 May 2020",
"name": "Anindo Majumdar",
"expertise": [
"Reviewer Expertise Noncommunicable 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\nSummary of the report:\nThe authors have conducted a timely and much needed research which was lacking even after about a decade of implementation of the NPCDCS. The use of mixed methods was required and quite rightly used for understanding such a complex process.\n\nThe main issue with the current manuscript looks to be a confusion between two research questions:\nWhat is the number and proportion eligible for screening of DM and HTN and subsequently, among those eligible, how many were screened, diagnosed and managed for the disease under routine programmatic field conditions?\n\nWhat effect does improving the existing documentation system have on the above mentioned variable and can the new system make documentation and reporting better?\n\nThe issue is, both are different research questions and trying to answer both of them in a single study will require better clarity in presentation and accepting the limitations of the study which will increase in number as a result of doing so. Nevertheless, since the issue is very important, with the incorporation of the suggested changes, the manuscript has the potential to be a very good one.\n\nFull report:\nSome specific issues are highlighted below:\n(A) Abstract\n\nHow is it a cohort design? There is no clear exposure. If we take age, gender and residence as exposure and outcome as screened for which analysis has been done, all these variables were collected at the same time for each participant with respect to the participants time. The quantitative part of this study has a cross-sectional design and should be mentioned as cross-sectional.\n\na) In results give denominator for 401. b) For the sentence - 'The documentation changes helped in identifying the eligible population' - this needs to be reworded to bring more clarity. c) Is willingness not a result of other factors such as recognition of relevance of screening rather being an independent enabler in itself?\n\n(B) Main manuscript:\nUnder Study Population subheading - How was the sample size for qualitative study reached at? What was the sampling technique used? These need to be mentioned.\n\nUnder subheading - Data variables, sources of data and data collection - While improving the system by developing better documentation systems has helped the researchers to get to the number screened, diagnosed and put on treatment, the qualitative part would not correctly reflect on the real field level functioning of the health centres under programmatic settings. For instance, some participants of in-depth interviews might liked to have mentioned the original documentation system (before improvement) as one of the main barriers. Since introducing the change in the recording system and seeing its outcome was not the objective, this should be mentioned as one of the limitations of the qualitative part.\n\nUnder data collection subheading - Were interns posted also taken as HCWs and interviewed?\n\nUnder Phase 2: Qualitative data collection subheading:\na) Since the PI himself interviewed the health workers of health centres, which he supports administratively, there is a definite element of bias while the HCWs would have given their views. Also, during the initial improvement of the documentation systems, the same stakeholders would have participated. It is quite possible they got motivated during this phase which would have had an impact on their responses. Also, they understood the study objectives during this phase, which would have also had an impact on their responses. This should be highlighted as a limitation.\n\nb) It is not clear how many KIIs and FGDs were conducted initially. This should be mentioned.\n\nUnder Statistical and data analysis subheading - Analysis should be as per cross-sectional study design and not cohort.\n\nIn Figure 2 - some statements are incomplete/incorrect: a) Care at PHC OPD - Initial part of the statement is missing. b) Diagnosed as HTN-8/63 - I think this should be 'not diagnosed as HTN'.\n\nUnder results section:\nQuantitative - Testing the association of socio-demographic characteristics with not being screened. Was this also a study objective?\nQualitative: a) “We have trained all staff including attendants on Glucometer usage. Therefore, the screening is going on smoothly despite staff shortage.”(HCP, 55 years, male) - Was this training given before the study or after they came to know about the study as a result of initial documentation improvement discussions? Since this would influence the results. Clarity is needed on this.\nb) Strength of PBS - It needs to be explained how this links to the study objectives that are related to opportunistic screening and not PBS.\nc) Patient-related enablers - Awareness of screening - Only persons who underwent screening have been interviewed. Since quantitative analysis was done to understand the factors associated with those 'not screened', it was equally important to know their perspective in qualitative interviews. This should be mentioned as a limitation.\nd) Documentation and reporting challenges - It needs to be explained more as the HCPs have also found the new reporting system as an enabler, although it would have made the new documentation/reporting format a bit more lengthy. This discrepancy needs to be explained well in the discussion part.\n\ne) Patient related barriers - Waiting time and inconvenience barrier and the reluctance for FBS barrier is contradictory to patient related enablers which has listed greater readiness to undergo screening and awareness and satisfaction with services. This needs to be explained in discussion section.\n\nf) In the limitations section - Also, asking the patient if the patient has already got DM/HTN is subject to wrong recoding of information if it was only based on verbal information from patients. Unless documentary evidence was sought to confirm, this should also be mentioned as a limitation.\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": "64187",
"date": "19 Jun 2020",
"name": "Subita P. Patil",
"expertise": [
"Reviewer Expertise Epidemiology",
"public health",
"preventive oncology",
"medical education"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is written very well.\n\nIMRAD format explained appropriately.\n\nThe design of the study is a good mix of qualitative & quantitative methods in current settings.\n\nThe conclusion, the strengths of the study are well narrated.\n\nThe outcome of the study is service-oriented for the benefit of the community.\n\nThe study can be accepted as it is.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "70384",
"date": "08 Oct 2020",
"name": "Mongal Singh Gurung",
"expertise": [
"Reviewer Expertise Noncommunicable Diseases (NCD) and Medical Statistics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very important operational research that provides insights for enhancing opportunistic screening and management of DM and HTN in Indian settings and other developing countries. Overall, the article is well written and merits indexing.\nI have the following suggestions/clarification:\nThe first sentence for results under the abstract could be split into two sentences for clarity and to segregate the findings by the study objectives. The second of the split sentences could present figures for cascades of screened, diagnosed, and initiated on treatment. The same comment applies to the HTN in the second sentence.\n\nAccording to the information provided under the study population section, it looks like all healthcare providers were interviewed but under Phase 2: Qualitative data collection section it says “… until information saturation was attained” as if recruitment of participants for the qualitative part were stopped when information saturation was attained.\" This may need clarification.\n\nHow many participants did not consent for audio recording? I suggest providing the exact number of participants who did not consent to the audio recording.\n\nProviding justification(s) for the choice of the KII and FGDs methods used may be useful for readers.\n\nHow many FGDs were conducted?\n\nIn Figure 2 the box “High Blood Pressure (SBP ≥140 and /or DBP ≥90) = 63/327(19.3%)” could be rephrased as “High Blood Pressure on screening (SBP ≥140 and /or DBP ≥90)=63/327(19.3%)” for clarity. Maybe the box “diagnosed as HTN =8/63(12.7%)” is “Not diagnosed as HTN =8/63(12.7%)”.\n\naRRs were calculated only for DM and not for HTN. Further, I think we adjust or control for other variables in the multiple variable models instead of “eliminate the confounders”. Only three variables (viz. age group, gender, and residence) were included in the models.\n\nThere were only 10 eligible individuals who were not screened for hypertension and only 2 women were not screened. This analysis may best be done away with. If Table 4 is retained then the last three columns of Table 4, which are empty and not relevant, could be removed.\n\nTable 2 shows information about the study participants, not all “persons aged ≥30 years seeking health care”. Therefore, the table title may need to be rephrased for clarity.\n\nGender and sex are very related words but they have different meanings. Usually, the administrative data of health is on sex rather than gender. Therefore, I suggest a review of its usage in the manuscript.\n\nNot including those who were not screened, in the qualitative interviews, to understand the barriers seems to be one of the limitations of this study.\n\nFew typos:\nThe was done to estimate the number of persons eligible for screening and to assess the feasibility of this strategy in such settings. A total of 6–8 participants were included in each FGD. FGDs. Of the 2697, 812 (30.2%), were aged 30–39 years with a mean age of 47.7 years (SD:12.3 years).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-335
|
https://f1000research.com/articles/8-1649/v1
|
16 Sep 19
|
{
"type": "Opinion Article",
"title": "How shall we treat locally advanced triple negative breast cancer?",
"authors": [
"Paulo Luz",
"David Dias",
"Ana Fortuna",
"Luis Bretes",
"Beatriz Gosalbez",
"David Dias",
"Ana Fortuna",
"Luis Bretes",
"Beatriz Gosalbez"
],
"abstract": "Triple negative breast cancer (TNBC) has been shown to respond to neoadjuvant chemotherapy (NACT). It has been established that achieving pathological complete response (pCR) for certain aggressive subtypes of breast cancer, including HER-2 (over-expressed) and TNBC, provides an important surrogate marker for predicting long-term clinical response and survival outcomes. How to increase the number of patients that achieve pCR remains challenging. Platinum-based NACT seems to be part of the solution and capecitabine, an active drug in metastatic breast cancer, but not a standard one in earlier stages may have found its place in the adjuvant setting.",
"keywords": [
"triple negative",
"breast cancer",
"neoadjuvant chemotherapy"
],
"content": "\n\nTriple negative breast cancer (TNBC) is immunohistochemically defined as the lack of expression of estrogen, progesterone receptor and human epidermal growth factor receptor 2 (HER-2). It accounts for 15–20% of breast cancer cases and is characterized as a molecular heterogeneous disease that usually presents an aggressive clinical behavior and higher prevalence in younger women1. Once TNBC has metastasized, it has the worst prognosis and the shortest OS of all breast cancer subtypes. On the other hand, TNBCs are highly chemo-sensitive and have been shown to respond very well to neoadjuvant chemotherapy (NACT)1–3.\n\nThe main goal of NACT strategies are not only to decrease the need for radical mastectomy but also to obtain an important predictive marker of favourable prognosis - the pathological complete response (pCR) - which is defined as the absence of invasive tumour cells (ypT0/is, ypN0/is). A correlation was observed between the pCR and the overall survival (OS) and disease free survival (DFS) outcomes in all subtypes of breast cancer, specially in aggressive ones such as HER2 positive and TNBC2. The pCR achieved by NACT represents to date the ideal endpoint in translational investigation of biomarkers and pharmacological treatments. Patients who do not have pCR after NACT with the combination of taxane and anthracycline have 20 to 30% risk of relapse2,3.\n\nHow to increase the number of patients that achieve pCR remains challenging. Platinum-based NACT seems to be part of the solution. Recently, a metanalysis that enrolled nine randomized clinical trials (RCT) with 2109 patients showed that platinum-based NACT compared to platinum-free NACT significantly increased pCR rate from 37.0% to 52.1%4. However, only two RCTs (CALGB 40603 and Gepar-Sixto trial) reported survival outcomes: no significant difference in event free survival (EFS) and OS was observed5,6. In the CALGB study adding carboplatin did not significantly impact survival. The absolute benefit in 3-year event-free survival of adding carboplatin was 4.9% and the OS differences were also not significant, with 81,9% OS in the carboplatin group versus 84,6% without carboplatin. These results conflict with those in the Gepar-Sixto trial. In the TNBC subgroup, carboplatin resulted in a significantly improved pCR rate over control (53% vs 37%; P = 0.005). This translated into an absolute benefit in 3-year event EFS for the addition of carboplatin over control of 9.7% (85.8% vs 76.1%, respectively). What reasons can explain this discrepancy? There are several differences between these two studies worth noting. GeparSixto had more-favorable baseline characteristics, as 26% of patients were cN0, compared with 42% in CALGB 40603. Additionally, a larger proportion were cT1 in GeparSixto (26% vs 11%). In CALGB 40603, the backbone therapy also included cyclophosphamide, which can also cause DNA damage like platinum agents, potentially making the treatment effect similar in the control and experimental arms5,6. Notably, BRCA-mutated patients experienced overall higher pCR rates, although no benefit was observed with the4.\n\nCurrent controversy in this field also includes the benefit of additional therapy after surgery. Given the still significantly high rates of residual disease after neoadjuvant therapy in TNBC, which portends inferior DFS, another approach to improving outcomes in this population is to add additional adjuvant therapy after surgery. The great majority of the studies focus on capecitabine, an active drug in metastatic TNBC. In the CREATE X trial (Capecitabine for Residual Cancer as Adjuvant Therapy)7 patients who have not achieved pathologic complete response after undergoing neoadjuvant therapy were randomized to receive standard treatment either with capecitabine or without (control). Among patients with triple negative disease, the rate of disease-free survival was 69.8% in the capecitabine group versus 56.1% in the control group, and the overall survival rate was 78.8% versus 70.3%7. Recently, at the San Antonio Breast Cancer Symposium 2018, the results of the phase III trial conducted by the Spanish group and the Central and South American group were presented, where treating patients with early-stage triple-negative breast cancer with capecitabine after surgery and standard chemotherapy did not significantly improve disease-free or overall survival compared with observation group8. One possible explanation for the discrepancy between the results of the CREATE-X trial and this trial may be the different prognostic features between the populations. The risk of relapse of the population was much lower than in the CREATE-X trial. So, capecitabine, an active drug in metastatic breast cancer, but not a standard one in earlier stages may have found its place.\n\nAn important question remains, should standard of care with NACT on TNBC rely on platinum-based treatments with the intent of achieving higher pCR rates and a probable benefit in OS or should it continue with taxane and antracyclin based combinations and consider the use capecitabine when pCR is not feasible?\n\nAccording to the authors´ perspectives, the impact of platinum on pCR and OS cannot simply be ignored. This option must be considered after balancing the potential benefits on disease outcomes versus increased toxicity. Special attention must be placed in older and frail patients that are still capable and willing to undergo NACT. In this subgroup of patients, taxane and antracyclin combination remains a valid first choice treatment and adjuvant capecitabine should be considered when residual tumor is still present. In young high risk patients who underwent platinum-based NACT and didn’t achieve pCR, adjuvant capecitabine should be discussed even we don’t have any RCT to support this idea.\n\nChemotherapy remains the backbone of TNBC, but research and development of new modalities of treatment continues. Recent and promising results were available in the metastatic setting. We hope that some of these drugs, like immunotherapy, may soon have a role in the neoadjuvant setting. Right now, we know neoadjuvant treatment allows us to re-write the story right at the start and we just cannot miss that opportunity.\n\n\nData availability\n\nNo data are associated with this study.",
"appendix": "References\n\nHarbeck N, Gnant M: Breast cancer. Lancet. 2017; 389(10074): 1134–1150. PubMed Abstract | Publisher Full Text\n\nCortazar P, Zhang L, Untch M, et al.: Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis. Lancet. 2014; 384(9938): 164–172. PubMed Abstract | Publisher Full Text\n\nvon Minckwitz G, Untch M, Blohmer JU, et al.: Definition and impact of pathologic complete response on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes. J Clin Oncol. 2012; 30(15): 1796–1804. PubMed Abstract | Publisher Full Text\n\nPoggio F, Bruzzone M, Ceppi M, et al.: Platinum-based neoadjuvant chemotherapy in triple-negative breast cancer: a systematic review and meta-analysis. Ann Oncol. 2018; 29(7): 1497–1508. PubMed Abstract | Publisher Full Text\n\nUntch M, Schneeweiss A, Salat C, et al.: 163PDLong-term survival analysis of the randomized phase II trial investigating the addition of carboplatin to neoadjuvant therapy for triple-negative (TNBC) and HER2-positive early breast cancer (GeparSixto). Ann Oncol. 2017; 28(suppl_ 5). Publisher Full Text\n\nSikov W, Berry D, Perou C, et al.: Abstract S2-05: Event-free and overall survival following neoadjuvant weekly paclitaxel and dose-dense AC +/- carboplatin and/or bevacizumab in triple-negative breast cancer: Outcomes from CALGB 40603 (Alliance). Cancer Res. 2016; 76(4 Supplement): S2-05-S2-05. Publisher Full Text\n\nMasuda N, Lee SJ, Ohtani S, et al.: Adjuvant Capecitabine for Breast Cancer after Preoperative Chemotherapy. N Engl J Med. 2017; 376(22): 2147–2159. PubMed Abstract | Publisher Full Text\n\nMartín M, Barrios CH, Torrecillas L, et al.: Abstract GS2-04: Efficacy results from CIBOMA/2004-01_GEICAM/2003-11 study: A randomized phase III trial assessing adjuvant capecitabine after standard chemotherapy for patients with early triple negative breast cancer. Cancer Res. 2019; 79(4 Supplement): GS2-04 LP-GS2-04. Publisher Full Text"
}
|
[
{
"id": "58166",
"date": "21 Jan 2020",
"name": "Brian D. Lehmann",
"expertise": [
"Reviewer Expertise Triple-negative breast cancer",
"translational research and genomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article the authors provide perspective on the use of platinum compounds in the treatment of early stage triple-negative breast cancer. The authors provide opinions on a recently published meta-analysis of nine randomized clinical trials (RCT) treated with platinum-based neoadjuvant chemotherapy.\nSpecific comments:\nPlease finish the sentence, “Notably, BRCA-mutated patients experienced overall higher pCR rates, although no benefit was observed with the.” The sentence and idea trying to be presented is incomplete.\n\nThe authors state, “What reasons can explain this discrepancy? There are several differences between these two-studies worth noting. GeparSixto had more-favorable baseline characteristics, as 26% of patients were cN0, compared with 42% in CALGB 40603.” This statement does not appear to be accurate. How is 26% of patients with cN0 in GeparSixto more favorable than 42% of patients with cN0 in CALGB? Please revise and double check the numbers. 42% is correct for CALGB as the patient population was limited to TNBC. GeparSixto included both TNBC and HER2 positive and at least in the combined population cN0 was 50%. Not sure what the value is when restricted to TNBC. The authors should consider comparing tumor grade, as higher grades are associated with better chemotherapy response. In CALGB 76% of patients had tumors of high grade, while 65% of tumors were high grade in GeparSixto, albeit in both TNBC and HER2.\n\nThe statement, “Recently, at the San Antonio Breast Cancer Symposium 2018…)” is not so recent anymore. Consider removing “recent” or replacing with newer published data.\n\nThe authors propose the very relevant question, “An important question remains, should standard of care with NACT on TNBC rely on platinum-based treatments with the intent of achieving higher pCR rates and a probable benefit in OS or should it continue with taxane and antracyclin based combinations and consider the use capecitabine when pCR is not feasible?” While the authors mention the data cannot be ignored, the authors do not mention how the question might finally be resolved. Especially given that there were no EFS or OS benefits from the two studies with published results, how can we justify the substantial toxicities of an additional platinum compound? Perhaps only when we see additional long-term analysis from the majority of RCTs with platinum compounds could the question be answered.\n\nThe authors conclude with, “We hope that some of these drugs, like immunotherapy, may soon have a role in the neoadjuvant setting.” While this may certainly be true in the metastatic setting, recent results suggest that this may not be the case in the neoadjuvant setting. Please include a discussion of immune checkpoint inhibitors in the neoadjuvant setting. Results from the NeoTRIPaPDL1 trial presented at SABC 2020 (Abstract #GS3-04) concluded that Atezolizumab with neoadjuvant chemotherapy does not improve pathologic complete response rates for patients with triple-negative breast cancer.\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": "5462",
"date": "04 May 2020",
"name": "Paulo Luz",
"role": "Author Response",
"response": "We appreciate the time and effort that you have dedicated to providing your valuable feedback on our manuscript. New evidence about immunotherapy in early TNBC is available (NeoTRIP and KEYNOTE 522). We incorporated that information in this new version of the article.We have also been able to incorporate changes to reflect most of the suggestions provided by you."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1649
|
https://f1000research.com/articles/9-322/v1
|
04 May 20
|
{
"type": "Study Protocol",
"title": "Impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria (mHealth-Cervix): Protocol for a randomised controlled trial",
"authors": [
"Kehinde S. Okunade",
"Omolola Salako",
"Adebola A. Adejimi",
"Oluwatosin J. Akinsola",
"Omolara Fatiregun",
"Muisi A. Adenekan",
"Olusanjo E. Moses",
"Bassey Ebenso",
"Matthew J. Allsop",
"Rose I. Anorlu",
"Jonathan S. Berek",
"Omolola Salako",
"Adebola A. Adejimi",
"Oluwatosin J. Akinsola",
"Omolara Fatiregun",
"Muisi A. Adenekan",
"Olusanjo E. Moses",
"Bassey Ebenso",
"Matthew J. Allsop",
"Rose I. Anorlu",
"Jonathan S. Berek"
],
"abstract": "Background: Incidence and mortality from cervical cancer have remained high due to many obstacles facing the implementation of organized screening programs in resource-constrained countries such as Nigeria. The application of mobile technologies (mHealth) to health services delivery has the potential to reduce inequalities, empower patients to control their health, and improve the cost-effectiveness of health care delivery. Aim: To assess the efficacy of mobile technology intervention on Pap test screening adherence compared to a control condition and also determine the factors affecting the uptake of Pap smear screening practices among women in Lagos. Methods: This is a multi-center randomized controlled trial that will involve women aged 25 to 65 years attending the General Outpatient clinics of the two tertiary health institutions in Lagos, Nigeria between April and December 2020. At baseline, a total of 200 National Health Insurance Scheme (NHIS) enrollees will be randomized to either a text message arm or usual care (control) arm. The primary outcome is the completion of a Pap smear within 6 months of enrolment in the study. The associations between any two groups of continuous variables will be tested using the independent sample t-test (normal distribution) or the Mann-Whitney U test (skewed data) and that of two groups of categorical variables with Chi-square X2or Fisher's exact test where appropriate. Using binary logistic regression model, we will adjust for age and other relevant sociodemographic and clinical variables and adherence to Pap test screening. Statistical significance will be defined as P-value less than 0.05. Discussion: The mHealth-Cervix study will evaluate the impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria as a way of contributing to the reduction in the wide disparities in cervical cancer incidence through early detection facilitated using health promotion to improve Pap smear screening adherence. Registration: PACTR202002753354517 13/02/2020",
"keywords": [
"Cervical cancer",
"Lagos",
"mHealth-Cervix",
"NHIS",
"Nigeria",
"Pap test"
],
"content": "Introduction\n\nCervical cancer is a major public health problem and is the fourth most common cancer in women worldwide accounting for an estimated 570,000 new cases annually1. More than 80% of the global burden of cervical cancer occurs in the less developed regions, where it accounts for almost 12% of all female malignancies2,3. In 2018, an estimated 311,000 deaths were attributed to cervical cancer, which accounts for 7.5% of all female cancer deaths with 70% of these occurring in developing countries1. Early detection of precursor lesions of cervical cancer through the use of screening tests has drastically reduced the incidence of the disease, especially in Western countries where Pap smear (cytology-based) screening has been introduced and now covers almost all eligible women4. However, in resource-constrained countries of the world such as Nigeria, cervical cancer incidence and mortality have remained high due to the many obstacles facing the implementation of organized screening programs5. A recent study conducted by our team among women in Lagos revealed Pap smear screening uptake of about 23% despite an awareness rate of 55%6.\n\nThe use of mobile technologies has increased exponentially in the last few years7. We reported in a study conducted in 2018 that mobile telephones could be found in 95% of households in Lagos and these were widely distributed across all socioeconomic classes8. Because of this increased access to technologies, mobile health (mHealth), or “medical and public health practice involving the use of mobile devices”9, has great potential in many health areas such as promotion and prevention10. The World Health Organization (WHO) report on mHealth in 20119 states that mobile health strategies exist in at least 75% of the countries in WHO regions. The application of mHealth to health services delivery has the potential to reduce inequalities, empower patients to control their health, and improve the cost-effectiveness of health care delivery11.\n\nAlthough still limited, there is growing evidence of success in the use of mobile phones for health (mHealth technologies) to support cancer prevention12–15. Nevertheless, there is currently no reported randomized controlled trial in sub-Saharan Africa (sSA) that examines the use of mobile health technologies in cancer prevention. This study will, therefore, aim to ascertain the efficacy of an intervention using mobile technologies on Pap smear screening adherence compared to standard care; and also determine the factors affecting the uptake of Pap smear screening services among women in Lagos, Nigeria. The introduction of mHealth, now regarded as one of the most promising investments for health in developing countries16, is a key innovative concept that will offer a unique opportunity for a paradigm shift in cervical cancer control in a resource-constrained setting like Nigeria and other parts of sSA.\n\n\nProtocol\n\nThis study (mHealth-Cervix) is a multi-center randomized controlled trial that will involve women aged 25 to 65 years who are attending the General Outpatient (GOP) clinics of two public tertiary health institutions in Lagos, Nigeria – Lagos University Teaching Hospital (LUTH), Idi-Araba and Lagos State University Teaching Hospital (LASUTH), Ikeja between April and December 2020. The trial protocol has been registered in the Pan African Clinical Trial Registry (PACTR) (PACTR202002753354517; version 3.0; February 13, 2020).\n\nLUTH and LASUTH are the two foremost public tertiary health institutions in Lagos that offer speciality and sub-speciality care including gynaecological oncology services such as Papanicolaou (Pap) test, human papillomavirus (HPV) testing and colposcopy for cervical cancer screening. The GOP clinics of the two hospitals run from Monday to Friday each week and attendees at the clinics are mostly enrollees of the National Health Insurance Scheme (NHIS) who have free cervical cancer screening as part of their health coverage. The hospitals act mainly as referral centers for other government-owned and private hospitals in Lagos State. Lagos State is the commercial capital of Nigeria which has a population of over 9 million inhabitants.\n\nEligible women are NHIS enrollees aged between 25 and 65 with no prior history of cervical cancer or cervical dysplasia; those not adherent with current recommendations for Pap smear screening (have not had a Pap smear within the last three years); those owning and using a personal cell-phone; those free from any mental or physical disabilities that inhibit them from understanding the implications of the study and those not considering relocating from their current residence within the next year. The exclusion criteria include women younger than 25 and older than 65 years old, those with history of cervical dysplasia and cancer, those who had a Pap test within the last three years, those with an ongoing pregnancy and those who refuse or withdraw consent during the study.\n\nInvestigators will identify eligible women from the GOP clinics of the two study sites on each day of the study period after which a 20–30 minutes educational health talk on cervical cancer and its prevention is given to these women by the clinic midwives with the assistance of the clinics’ health care teams as part of their usual standard of care. Eligible women are then invited by the investigators to give consent for participation in the clinical trial upon explanation of the purpose and nature of the study. Once consent is obtained, an interviewer-administered questionnaire (see extended data17) is then used to obtain baseline information on sociodemographic variables, cell-phone use, and distance of participants’ residence from the clinics (measured in kilometer using the Google maps). Each woman will receive an estimated $5 credit charge on their cellphones as a token to enable the investigators to keep their phone numbers for as long as the intervention lasts. The women in both arms are each given the complementary card containing the cell-phone number of the cytology clinic coordinators (who are also part of the study) and are subsequently scheduled for 6-month follow-up appointments from their dates of study enrolment. After follow-up, data will be collected from each woman on completion of Pap test screening, the number of GOP clinics attended, the interval from enrollment to date of Pap smear testing and functionality of cell-phone during the same follow-up period (that is, if the cell-phone was in use for at least 5 out the 6 months follow-up or not).\n\nFollowing the baseline assessment, enrolled women will be randomized to either a text message (intervention) arm or usual care (control) arm using computer-generated block randomization codes using Random Allocation software version 1.0 (May 2004) by a study statistician. Investigators and statisticians will be blinded to the allocation groups [Figure 1]. The allocation sequence is concealed in sealed opaque envelopes that will be kept in a locked file cabinet at each of the study sites until participants assignment is completed.\n\nIntervention (mHealth) arm – We chose Nexmo® as the platform to deliver the mHealth messages given its low cost and reliability. Participants will be sent messages containing information and encouragement to undergo cervical cancer screening. Information will also be provided about the Cytology clinic hours, contact information and locations in the two participating hospitals. Information and motivational text messages will be delivered twice monthly for the next 6-months after enrollment.\n\nUsual care (control) arm – Participants randomized to the control arm will not receive additional reminder messages from study staff other than the usual care received at enrollment. Usual care consists of any information on Pap tests and cervical cancer risk reduction typically provided by midwives to all women at the clinics as will be done at enrollment. These women can then schedule an appointment for their Pap smear testing in person at the GOP or Cytology clinics at any time during the 6-month follow-up period.\n\nPrimary endpoint – This is the completion of a Pap smear within 6 months of enrollment in the study. Participants will be tracked via medical record review as well as through phone calls at 6-month after their enrolment.\n\nSecondary endpoint – This is an assessment of the various factors affecting Pap smear uptake or adherence within 6-month of enrollment among the participating women.\n\nSample size – We calculated sample size using G*Power for Windows version 3.1.9.2 (Kiel University, Germany). Using data from our published study6, we estimated the proportion of Pap smear adherence to be approximately 23% for the usual care (control) arm and 60% for the text message (intervention) arm. This is powered for a two-sided test with a Type I error rate of 5% and 80% power, i.e. Zα=1.96 and Zβ=0.84, adjusted for a 10% attrition rate. Therefore, the power calculations will be based on a sample size of 100 in each of the two study arms (making a total sample size of 200).\n\nStatistical analysis – The intention-to-treat principle will be used in the final analyses. Statistical analyses will be carried out using SPSS version 23.0 for Windows (Armonk, NY: IBM Corp.). Pap smear adherence will be coded as a binary variable. The intervention will be evaluated based only on Pap smear completion at 6-month after the enrollment and baseline survey. The associations between any two groups of continuous variables will be tested using the independent sample t-test (normal distribution) or the Mann-Whitney U test (skewed data) and that of two groups of categorical variables with Chi-square (χ2) or Fisher's exact test where appropriate. Using binary logistic regression analysis, we will adjust for age, socioeconomic class, parity, marital status, number of GOP clinic attendance, the distance of residence from the clinics, interval from enrollment to date of Pap smear testing, the functionality of cellphones and other relevant demographic and clinical variables. Statistical significance will be defined as P-value less than 0.05.\n\nAll investigators and research assistants will be required to undergo training before the trial to guarantee consistent practice. The training will include an understanding of inclusion/exclusion criteria, follow-up procedures, and completion of the questionnaire. Identifiable data will be transferred to an electronic database system located in a guarded facility at the trial site by the research assistant. Access to identifiable data is restricted only to the PI during and after the trial completion. The trial will be monitored by quality assurance personnel from the research management office of the College of Medicine, University of Lagos, who will be independent of the study team, and an independent steering committee. There will be periodic monitoring to guarantee accuracy and quality throughout the study period. The essential documents (consent information, enrolment, protocol deviations, and losses to follow-up) will be monitored and checked for accuracy and completeness by the monitors. The principal investigator (KSO) is responsible for the overall project and for organizing steering committee meetings. The independent steering committee will be responsible for ensuring the overall safety of participants, coordinating study meetings, supervising the study, monitoring data safety, and overseeing quality control.\n\nThis study protocol was approved by the Health Research Ethics Committee of College of Medicine, University of Lagos (CMUL HREC) on 2nd February 2020 with approval number CMUL/HREC/12/19/704. Participants will provide written informed consent before their participation in the trial and the participant will keep a signed copy of the consent form for future references.\n\nThe trial will be reported in line with the Consolidated Standards of Reporting Trials (CONSORT) checklist and the results will be disseminated in peer-review scientific journals. Important modifications to the trial protocol will be communicated to the funder, study investigators, CMUL HREC, trial participants and trial registries.\n\nAt the time of initial manuscript submission, recruitment is yet to commence into the trial. Participants’ enrolment will start in May 2020, and the last woman is expected to be included in the trial in July 2020. The manuscript reports protocol version 3.0 (30th March 2020).\n\n\nDiscussion\n\nCervical cancer is a major public health disease in Nigeria. However, screening is still not a national priority as only 23% of adult females has ever been screened6. There is wide use of mobile technologies in Nigeria and most parts of sub-Saharan Africa, but there are currently no randomized controlled trials that have described the use of these technologies for cancer screening. This protocol describes a randomised controlled trial of a novel intervention (mHealth technologies using text messages) to improve Pap smear screening adherence among women in Lagos. The mHealth-Cervix study will evaluate the impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria and determine the factors that affect this screening adherence as a means of reducing the disparities in the incidence of cervical cancer through early detection facilitated using health promotion to improve Pap smear screening adherence. If found to be effective in increasing adherence to screening, the mHealth intervention strategy may become an important tool for reducing the cervical cancer burden, and its associated morbidity and mortality. This could also be applied in the future for the health promotion and prevention of other major disease conditions.\n\n\nData availability\n\nNo data are associated with this article\n\nHarvard Dataverse: Replication Data for: Impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria (mHealth-Cervix): Protocol for a randomised controlled trial https://doi.org/10.7910/DVN/GXE1ON17\n\nThis trial protocol contains the following underlying data:\n\nConsent form.pdf (Study consent form)\n\nQuestionnaire_f1000.docx\n\nSPIRIT checklist for “Impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria (mHealth-Cervix): Protocol for a randomised controlled trial” https://doi.org/10.7910/DVN/GXE1ON17\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 appreciate the staff of the Research Management Office (RMO) of the College of Medicine, University of Lagos for their assistance in securing funding for this study.\n\n\nReferences\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global Cancer Statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394–424. PubMed Abstract | Publisher Full Text\n\nSankaranarayanan R: Overview of Cervical Cancer in the Developing World. Int J Gynecol Obstet. 2006; 95(Suppl 1): S205–S210. PubMed Abstract | Publisher Full Text\n\nSankaranarayanan R, Nessa A, Esmy PO, et al.: Visual inspection methods for cervical cancer prevention. Best Prac Res Clin Obstet Gynaecol. 2012; 26(2): 221–32. PubMed Abstract | Publisher Full Text\n\nJordan J, Arbyn M, Martin-Hirsch P, et al.: European guidelines for quality assurance in cervical cancer screening: recommendations for clinical management of abnormal cervical cytology, part 1. Cytopathology. 2008; 19(6): 342–54. PubMed Abstract | Publisher Full Text\n\nPapillomavirus H. Related Cancers in Cameroon. Summary Report, WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). 2010.\n\nOkunowo AA, Daramola ES, Soibi-Harry AP, et al.: Women's knowledge of cervical cancer and uptake of Pap smear testing and the factors influencing it in a Nigerian tertiary hospital. Journal of Cancer Research and Practice. 2018; 5(3): 105–11. Publisher Full Text\n\nThe World in 2014. ICT facts and figures. ITU world telecommunications. Retrieved June 4, 2014. Reference Source\n\nOkunade KS, Salako O, Adenekan M, et al.: The uptake of cervical cancer control services at a cancer information service center in Lagos, Nigeria. Niger J Gen Pract. 2018; 16: 20–4. Publisher Full Text\n\nMHealth: New Horizons for Health through Mobile Technologies. Global Observatory for eHealth Series, 2011; 3: Retrieved June 4, 2014Reference Source\n\nAylward D, Leão B, Curioso W, et al.: Can you heal me now? Potential (and pitfalls) of mHealth. Americas Quarterly. 2010; 4(3): 88–95. Reference Source\n\nGreen paper on mobile health (mHealth). European Commission; 2010; Retrieved June 4, 2014 Reference Source\n\nKällander K, Tibenderana JK, Akpogheneta OJ, et al.: Mobile health (mHealth) approaches and lessons for increased performance and retention of community health workers in low- and middle-income countries: a review. J Med Internet Res. 2013; 15(1): e17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalako O, Robert AA, Okunade KS, et al.: Utilization of Cancer Information System for Breast Cancer Control in Lagos, Nigeria. Pan Afr Med J. 2016; 24: 323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUmali E, McCool J, Whittaker R: Possibilities and Expectations for mHealth in the Pacific Islands: Insights From Key Informants. JMIR mHealth uHealth. 2016; 4(1): e9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuinley KE, Gormley RH, Ratcliffe SJ, et al.: Use of mobile telemedicine for cervical cancer screening. J Telemed Telecare. 2011; 17(4): 203–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCurioso WH, Mechael PN: Enhancing 'M-health' with south-to-south collaborations. Health Aff (Millwood). 2010; 29(2): 264–7. PubMed Abstract | Publisher Full Text\n\nOkunade KS, Salako O, Adejimi A, et al.: \"Impact of mobile technologies on cervical cancer screening practices in Lagos, Nigeria (mHealth-Cervix): Protocol for a randomised controlled trial\". 2020; Harvard Dataverse, V3. http://www.doi.org/10.7910/DVN/GXE1ON"
}
|
[
{
"id": "63099",
"date": "13 May 2020",
"name": "Adeola Olaitan",
"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 research addresses the importance of improving compliance with cervical screening using mobile phone technology. I am not familiar with the platform described and would suggest another reviewer comments on the appropriateness.\n\nThis manuscript presents methods but no results yet. I suggest the paper will make for more interesting reading when results are available. Also the endpoints of the study are attendance for screening. As the purpose of screening is to prevent cervical cancer, a more important goal will be difference in outcome between the two study groups in terms of detection rates of premalignant lesions and eventual outcome. I appreciate the numbers may be too small to demonstrate a reduction in cervical cancer.\n\nFinally I question why cervical smears are the chosen screening methods when all available data indicate that HPV testing is more reliable and objective and is indeed the method of choice in the national cancer plan.\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? Partly",
"responses": []
},
{
"id": "63096",
"date": "01 Jun 2020",
"name": "Lateef Akinola",
"expertise": [
"Reviewer Expertise Obstetrics and gynaecology",
"reproductive medicine and surgery specialist"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 'Impact of technologies on cervical cancer screening practices in Lagos, Nigeria (mHealth Cervix): Protocol for a RCT by Okunade KS et al., set out to understudy a very relevant and timely aspect on cancer preventive initiative in Nigeria specifically in Lagos the economic hub and most populous city of Nigeria. This is aimed at preventing the incidence and mortality from cervical cancer in the urban city of Lagos in Nigeria. The methodology to study MHealth Cervix in Lagos is excellent and will cover the relevant population of Lagos city. The power 80% is significant enough and the sample size is robust. The statistical analysis package is more than adequate to analyse the research result specifically by using the two tests for comparison of means or test of significance. The research report is anticipated and I hope the author will proceed to the more predictive test using HPV screening in their future research on this very important subject matter.\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/9-322
|
https://f1000research.com/articles/7-1310/v1
|
17 Aug 18
|
{
"type": "Research Article",
"title": "First draft genome assembly of the Argane tree (Argania spinosa)",
"authors": [
"Slimane Khayi",
"Nour Elhouda Azza",
"Fatima Gaboun",
"Stacy Pirro",
"Oussama Badad",
"M. Gonzalo Claros",
"David A. Lightfoot",
"Turgay Unver",
"Bouchra Chaouni",
"Redouane Merrouch",
"Bouchra Rahim",
"Soumaya Essayeh",
"Matike Ganoudi",
"Rabha Abdelwahd",
"Ghizlane Diria",
"Meriem Alaoui Mdarhi",
"Mustapha Labhilili",
"Driss Iraqi",
"Jamila Mouhaddab",
"Hayat Sedrati",
"Majid Memari",
"Noureddine Hamamouch",
"Juan de Dios Alché",
"Noureddine Boukhatem",
"Rachid Mrabet",
"Rachid Dahan",
"Adelkhaleq Legssyer",
"Mohamed Khalfaoui",
"Mohamed Badraoui",
"Yves Van de Peer",
"Tatiana Tatusova",
"Abdelhamid El Mousadik",
"Rachid Mentag",
"Hassan Ghazal",
"Slimane Khayi",
"Nour Elhouda Azza",
"Fatima Gaboun",
"Stacy Pirro",
"Oussama Badad",
"M. Gonzalo Claros",
"David A. Lightfoot",
"Turgay Unver",
"Bouchra Chaouni",
"Redouane Merrouch",
"Bouchra Rahim",
"Soumaya Essayeh",
"Matike Ganoudi",
"Rabha Abdelwahd",
"Ghizlane Diria",
"Meriem Alaoui Mdarhi",
"Mustapha Labhilili",
"Driss Iraqi",
"Jamila Mouhaddab",
"Hayat Sedrati",
"Majid Memari",
"Noureddine Hamamouch",
"Juan de Dios Alché",
"Noureddine Boukhatem",
"Rachid Mrabet",
"Rachid Dahan",
"Adelkhaleq Legssyer",
"Mohamed Khalfaoui",
"Mohamed Badraoui",
"Yves Van de Peer",
"Tatiana Tatusova",
"Abdelhamid El Mousadik"
],
"abstract": "Background: The Argane tree (Argania spinosa L. Skeels) is an endemic tree of southwestern Morocco that plays an important socioeconomic and ecologic role for a dense human population in an arid zone. Several studies confirmed the importance of this species as a food and feed source and as a resource for both pharmaceutical and cosmetic compounds. Unfortunately, the argane tree ecosystem is facing significant threats from environmental changes (global warming, over-population) and over-exploitation. Limited research has been conducted, however, on argane tree genetics and genomics, which hinders its conservation and genetic improvement. Methods: Here, we present a draft genome assembly of A. spinosa. A reliable reference genome of A. spinosa was created using a hybrid de novo assembly approach combining short and long sequencing reads. Results: In total, 144 Gb Illumina HiSeq reads and 7.2 Gb PacBio reads were produced and assembled. The final draft genome comprises 75 327 scaffolds totaling 671 Mb with an N50 of 49 916 kb. The draft assembly is close to the genome size estimated by k-mers distribution and covers 89% of complete and 4.3 % of partial Arabidopsis orthologous groups in BUSCO. Conclusion: The A. spinosa genome will be useful for assessing biodiversity leading to efficient conservation of this endangered endemic tree. Furthermore, the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes.",
"keywords": [
"Argane",
"Argania spinosa",
"Endemic",
"Genome",
"Assembly",
"Morocco",
"International Argane Genome Consortium"
],
"content": "Introduction\n\nArgania spinosa (L. Skeels) is a tree endemic to the South West of Morocco and occupying arid and semi-arid regions totaling up to around 900,000 ha1. The argane tree forest was recognized as biosphere reserve (Arganeraie Biosphere Reserve) by UNESCO in 19982. It is the only unique member of the tropical Sapotaceae family in Morocco3. In addition to its ecological role in preventing soil erosion and desertification, the argane tree has great cultural and socio-economic importance. The oil extracted from the seed is considered the most expensive edible oil in the world with great cosmetic value and therapeutic potential4–6. Argane oil represents a significant source of dietary fatty acids, while the Argane fruit is used as livestock feed by the local population7–9. Phytochemical composition of Argane fruits reveals different classes of bioactive compounds, including essential oils, fatty acids, triacylglycerols, flavonoids and their acylglycosyl derivatives, monophenols, phenolic acids, cinnamic acids, saponins, triterpenes, phytosterols, ubiquinone, melatonin, new aminophenols, and vitamin E. Argane oil contains high levels of antioxidant compounds. The long-chain fatty acids in Argane oil are primarily represented by unsaturated oleic acid, then linoleic acid, palmitic acid and stearic acid10.\n\nThe distribution area of the Argane forest decreased drastically during the 18th century. Furthermore, about 44 % of the forest was again lost between 1970 and 2007. While there are multiple causes, desertification and overgrazing form the main pressures on the Argane forest11,12. Therefore, the management and conservation of the remaining genetic resources of Argane forest are urgent priorities. In recent decades, several studies have been conducted to evaluate the genetic diversity of the Argane tree using morphological13,14, chemical10,15, biochemical6,16 and standard molecular marker techniques, all with the aim of describing the genetic diversity of Argane trees and addressing ecological and conservation issues17–25. The karyotype of A. spinosa (L.) is constituted of ten pairs of chromosomes (2n =2x =20)3. Until now, no reference genome was available of the A. spinosa species. Here, we present the Argane tree genome assembled from short and long DNA reads using a hybrid assembly strategy.\n\n\nMethods and results\n\nThe Argane tree (Argania spinosa, taxid 85883, Sapotaceae, family, order Ericales), named Argane AMGHAR, to be sequenced was selected for its biological and ecological characteristics (Figure 1). This was a 9-year-old shrub, with weeping form (geotropic, unlike the erect have) with only one main trunk 3 m in height. The ripe fruits has a rounded shape. The plant had semi-evergreen dwarf leaves. The shrub is native to the valley of the plain of Sous, an arid climate with an annual average rainfall of around 220 mm, located between the hills of the Anti-Atlas towards the South East, the Western High Atlas towards the North-West and the Atlantic Ocean towards the West (9°32′ 00″N, 30°24′ 00″W; Altitude: 126 m).\n\n(A) Map of Sous region with Argania species distribution. (B) Picture of the tree # Argane Amghar. (Photograph taken by A. El Mousadik).\n\nGenomic DNA was extracted from lyophilized leaf tissues of a single tree (Argane AMGHAR) using the Plant DNeasy mini kit (Qiagen, USA). The Argane tree genome was shotgun-sequenced using both PacbioTM (Menlo Park, CA, USA) and IlluminaTM (San Diego, CA, USA) sequencing technologies, generating 7.2 Gb and 144 Gb of data, respectively. A gel-based size selection of DNA was performed for fragments ≥ 2 kb. Paired-end libraries with average insert sizes of 600 bp were constructed with NexteraTM DNA Library Prep Kit for Illumina (New England BiolabsTM, New Brunswick, MA, USA). These libraries were sequenced on an Illumina HiSeq XTen platform using the PE-150 module and yielded 957,451,810 reads (Table 1). These data was trimmed of adapters and low-quality sequences, yielding a clean set of 936,053,040 reads, representing 160× genome coverage, assuming a genome size of 573 Mb as estimated by the k-mer frequency analysis (described below). Raw reads were deposited at the NCBI Sequence Read Archive (SRA) under accession numbers: SRX3207155 and SRX3207156, corresponding to two independent runs from the same plant DNA sample. In addition, single-molecule long reads from the PacBio RS II platform (Pacific Biosciences, USA) were used to assist the subsequent de novo genome assembly using Illumina. Genomic sequencing libraries were constructed using the PacBio DNA template preparation kit 2.0 (Pacific Biosciences of California, Inc., Menlo Park, CA) for SMRT sequencing on the PacBio RS II machine (Pacific Biosciences of California, Inc.) according to the manufacturer's instructions, with a size range of 2-15 kb. The constructed libraries were sequenced on six SMRT cells on a PacBio RSII sequencer. The sequences of the 6 SMRT cell runs were deposited at the NCBI SRA under accession numbers: SRX1898029/SRX1898030/SRX1898031/SRX1898032/SRX1898033/SRX1898034. The sequencing runs produced about 7.2 Gb, consisting of 6,705,437 reads with an average read length of 2.5 kb and representing about 12× genome coverage, again assuming a genome size of 573 Mb (Table 1).\n\nQuality-filtered reads from the Illumina platform were subjected to k-mer frequency distribution analysis with JELLYFISH v2.1.4 software26,27. Analysis parameters were set at -k 21 and 25, and the final result was plotted as a frequency graph (Figure 2). Two distinctive modes were observed from the distribution curve: the higher peak at a depth of 44 and reflecting the high heterozygosity of the Argane genome; the lower peak provided a peak depth of 87 for the estimation of the genome size28. Based on the total number of k-mers obtained, the Argane genome size was calculated to be approximately 573 Mb and 615 Mb, for 21- and 25-mers respectively, using the following formula: total number of k-mer / Peak depth. The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % (Figure 2). The estimated genome size seems to be credible compared to the ones of four other Sapotaceae family members. In fact, according to the Plant DNA c-values Database, the genome sizes of these four species ranged from 273 Mb in Mimusops elengi L. (c = 0.28 pg) to 2,513 Mb in Isonandra villosa L. (c = 2.57 pg). The other two species are Planchonella eerwah (c = 0.54 pg, 528 Mb) and Madhuca longifolia (c = 0.99 pg, 968 Mb).\n\nPrior to assembly, Illumina and PacBio raw reads were trimmed for quality and adaptor removal using bbduk.sh from BBmap suite (https://github.com/BioInfoTools/BBMap). Short and long reads were assembled following a hybrid approach using MaSuRCA assembler v.3.2.229. The initial assembly consists of 671,690,540 bp composed of 82,183 contigs with the largest size being 422,848 bp and an N50 of 43,654 bp. The very few contigs (8) with length less than 200 bp were filtered out and the remaining contigs were scaffolded into 75,327 scaffolds totaling 670,096,797 bp; the N50 reached 49,916 bp and the assembly accounted for 2,982,868 Ns with 445.14 Ns per 100 kb (Table 2). The scaffolding was done using initial contigs and implemented in MaSuRCA v3.2.4 assembler script using Celera Assembler v8.3. The GC content was estimated to be 33%. The assembly was screened by VecScreen to look for and remove remaining vector contamination. Based on the VecSreen report, contigs containing mitochondrial/chloroplast were also removed. Trimmed PE reads were mapped on the final assembly using CLC genomics (v11.0, CLCbio, Arhus, Denmark) with 0.8 in length and 0.9 in sequence similarity. In total, 94% of the reads were mapped against the Argane genome. The 6% reads that were unmapped may result from the stringency of mapping criteria used.\n\nN50 size defined as the value N such that at least 50% of the genome is covered by scaffolds of size N or larger.\n\nThe difference between the genome size estimation and assembly size may be due to the use of parameters excluding extremely high frequency k-mers. They often represent organelle sequences, eventual contaminants inflating the genome size estimation30, or the high-frequency of repetitive regions found in plant genomes. Furthermore, the genome is highly heterozygous and different allelic regions would inflate assembly size. To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with Arabidopsis lineage-specific orthologous groups31, which showed that the assembly contained 89% (1271 genes) of complete and 4.3% (62 genes) of partial sequences that were Arabidopsis orthologs.\n\n\nConclusions\n\nThis draft genome assembly is a first step towards a global and integrative omics strategy for exhaustive characterization of the Argane tree. In particular, future work will focus on structurally annotating the genome using predictive tools and transcriptome analysis. Other future work will focus on functional gene annotation, finding evidence for genome duplication and comparative genome evolution. A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing. The metabolome, and analysis of Argane oil biosynthesis, as well as the tree’s microbiome should also be analyzed. To this end, and in order to coordinate the strong interests of the Plant Genomics community for this precious tree, the International Argane Genome Consortium (IAGC) and a resource website has been created (www.arganome.org).\n\n\nData availability\n\nAll of the A. spinosa datasets can be retrieved under BioProject accession number PRJNA294096: http://identifiers.org/bioproject:PRJNA294096. The raw reads are available at NCBI Sequence Reads Archive under accession number SRP077839: http://identifiers.org/insdc.sra:SRP077839. The complete genome sequence assembly project has been deposited at GenBank under accession number QLOD00000000: http://identifiers.org/ncbigi/GI:1408199612. Data can also be retrieved via the International Argane Genome Consortium (IAGC) website: http://www.arganome.org.\n\n\nAuthor information\n\nSlimane Khayi and Nour Elhouda Azza are co-first authors; Rachid Mentag and Hassan Ghazal contributed equally as supervisors.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Iridian Genome Foundation (MD, USA). H.G. is supported by a Grant from the NIH (MD, USA) for H3ABioNet/H3Africa (grant numbers U41HG006941 and U24 HG006941-06). O.B. and B.C. are Fulbright JSD (USA) grant recipients. This work also benefited from support of Midterm Research Program of INRA-Morocco through the use of its bioinformatics platform.\n\n\nAcknowledgements\n\nThanks are due to the Fulbright Program for supporting Morocco to US exchange PhD students. We would like to thank Lieven Sterck for a critical reading of the manuscript.\n\n\nReferences\n\nLefhaili A: FAO Forest Resources Assessment: Morocco Country Report. Rome. FAO; 2010. Reference Source\n\nUNESCO - MAB Biosphere Reserves Directory. 2018. Reference Source\n\nMajourhat K, Jabbar Y, Araneda L, et al.: Karyotype characterization of Argania spinosa (L.) Skeel (Sapotaceae). South Afr J Bot. 2007; 73(4): 661–3. Publisher Full Text\n\nMonfalouti HE, Guillaume D, Denhez C, et al.: Therapeutic potential of argan oil: a review. J Pharm Pharmacol. 2010; 62(12): 1669–1675. PubMed Abstract | Publisher Full Text\n\nKhallouki F, Spiegelhalder B, Bartsch H, et al.: Secondary metabolites of the argan tree (Morocco) may have disease prevention properties. Afr J Biotechnol. 2005; 4(5): 381–388. Reference Source\n\nKhallouki F, Younos C, Soulimani R, et al.: Consumption of argan oil (Morocco) with its unique profile of fatty acids, tocopherols, squalene, sterols and phenolic compounds should confer valuable cancer chemopreventive effects. Eur J Cancer Prev. 2003; 12(1): 67–75. PubMed Abstract | Publisher Full Text\n\nCharrouf Z, Guillaume D: Argan oil: Occurrence, composition and impact on human health. Eur J Lipid Sci Technol. 2008; 110(7): 632–6. Publisher Full Text\n\nLybbert TJ, Aboudrare A, Chaloud D, et al.: Booming markets for Moroccan argan oil appear to benefit some rural households while threatening the endemic argan forest. Proc Natl Acad Sci. 2011; 108(34): 13963–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlba-Sánchez F, López-Sáez JA, Nieto-Lugilde D, et al.: Long-term climate forcings to assess vulnerability in North Africa dry argan woodlands. Rocchini D, editor. Appl Veg Sci. 2015; 18: 283–96. Publisher Full Text\n\nKhallouki F, Voggel J, Breuer A, et al.: Comparison of the major polyphenols in mature Argan fruits from two regions of Morocco. Food Chem. 2017; 221: 1034–1040. PubMed Abstract | Publisher Full Text\n\nle Polain de Waroux Y, Lambin EF: Monitoring degradation in arid and semi-arid forests and woodlands: The case of the argan woodlands (Morocco). Appl Geogr. 2012; 32(2): 777–86. Publisher Full Text\n\nMcGregor HV, Dupont L, Stuut JBW, et al.: Vegetation change, goats, and religion: a 2000-year history of land use in southern Morocco. Quat Sci Rev. 2009; 28(15–16): 1434–48. Publisher Full Text\n\nAit Aabd N, El Ayadi F, Ms F: Evaluation of agromorphological variability of argan tree under different environmental conditions in Morocco: implication for selection. Int J Biodivers Conserv. 2011; 3(3): 73–82. Reference Source\n\nBani-Aameur F, Ferradous A: Fruits and stone variability in three argan (Argania spinosa (L.) Skeels) populations. Int J For Genet. 2001; 8: 39–45. Reference Source\n\nKhallouki F, Haubner R, Ricarte I, et al.: Identification of polyphenolic compounds in the flesh of Argan (Morocco) fruits. Food Chem. 2015; 179: 191–198. PubMed Abstract | Publisher Full Text\n\nKlika KD, Khallouki F, Owen RW: Amino phenolics from the fruit of the argan tree Argania spinosa (Skeels L.). Z Naturforsch C. 2014; 69(9–10): 363–367. PubMed Abstract | Publisher Full Text\n\nPakhrou O, Medraoui L, Yatrib C, et al.: Assessment of genetic diversity and population structure of an endemic Moroccan tree (Argania spinosa L.) based in IRAP and ISSR markers and implications for conservation. Physiol Mol Biol Plants. 2017; 23(3): 651–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl Bahloul Y, Dauchot N, Machtoun I, et al.: Development and characterization of microsatellite loci for the Moroccan endemic endangered species Argania spinosa (Sapotaceae). Appl Plant Sci. 2014; 2(4): pii: apps.1300071. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakhchar A, Haworth M, El Modafar C, et al.: An Assessment of Genetic Diversity and Drought Tolerance in Argan Tree (Argania spinosa) Populations: Potential for the Development of Improved Drought Tolerance. Front Plant Sci. 2017; 8: 276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajourhat K, Jabbar Y, Hafidi A, et al.: Molecular characterization and genetic relationships among most common identified morphotypes of critically endangered rare Moroccan species Argania spinosa (Sapotaceae) using RAPD and SSR markers. Ann For Sci. 2008; 65(8): 805–805. Publisher Full Text\n\nEl Mousadik A, Petit RJ: Chloroplast DNA phylogeography of the argan tree of Morocco. Mol Ecol. 1996a; 5(4): 547–555. PubMed Abstract | Publisher Full Text\n\nEl Mousadik A, Petit RJ: High level of genetic differentiation for allelic richness among populations of the argan tree [Argania spinosa (L.) Skeels] endemic to Morocco. Theor Appl Genet. 1996b; 92(7): 832–839. PubMed Abstract | Publisher Full Text\n\nMouhaddab J, Ait Aabd N, Achtak H, et al.: A Patterns of Genetic Diversity and Structure at Fine Scale of an Endangered Moroccan Endemic Tree (Argania spinosa L. Skeels) Based on ISSR Polymorphism. Notulae Botanicae Horti Agrobotanici Cluj- Napoca. 2015; 43(2): 528–535. Reference Source\n\nMouhaddab J, Ait Aabd N, Msanda F, et al.: Assessing genetic diversity and constructing a core collection of an endangered Moroccan endemic tree [Argania spinosa (L.) Skeels]. Moroccan J Biol. 2017; 13: 1–12. Reference Source\n\nMouhaddaba J, Msandaa F, Filali-Maltoufb A, et al.: Using microsatellite markers to map genetic diversity and population structure of an endangered Moroccan endemic tree (Argania spinosa L. Skeels) and development of a core collection. Plant Gene. 2017; 10: 51–59. Publisher Full Text\n\nLiu B, Shi Y, Yuan J, et al.: Estimation of genomic characteristics by analyzing k-mer frequency in de novo genome projects. ArXiv13082012 Q-Bio. 2013. Reference Source\n\nMarçais G, Kingsford C: A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27(6): 764–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKajitani R, Toshimoto K, Noguchi H, et al.: Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 2014; 24(8): 1384–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimin AV, Marçais G, Puiu D, et al.: The MaSuRCA genome assembler. Bioinformatics. 2013; 29(21): 2669–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVurture GW, Sedlazeck FJ, Nattestad M, et al.: GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics. 2017; 33(14): 2202–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–2. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "38275",
"date": "17 Sep 2018",
"name": "Amit Sinha",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current manuscript, the authors present the first draft genome assembly for the argan tree Argania spinosa, a tree with significant ecological as well as commercial importance. The authors have used a hybrid assembly approach, combing data from both Illumina short-reads and PacBio long-reads technology. Use of such hybrid approaches is welcome, as they can combine the advantages offered by different technological platforms. Based on a k-mer analysis, the authors also provide a reasonable estimate of the genome-size and the level of heterozygosity. These are important metrics to report, as they can guide the methods to be used for genome assembly, as well as for interpretation of the assembled genome sequence. The draft genome has been satisfactorily assembled, using state of the art computational methods, and it contains 89% of the predicted core genes as defined in the BUSCO software.\nIt is hoped that in their future studies, the authors will follow up with more improvements to the draft genome, including gene annotations using transcriptomic data.\nOverall, manuscript provides valuable information and sequence resources that will be useful to the scientific community. I have only 1 major and 6 minor comments/suggestions that I think the authors should be able to address.\nMajor reservation:\n1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for NOT using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript.\nMinor comment/suggestion:\n1. For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence (CCS) reads?\n2. The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ?\n3. The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and a estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 = 236X. Please correct or explain further.\n4. In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided.\n5. For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined.\n6. The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "4751",
"date": "04 May 2020",
"name": "Hassan Ghazal",
"role": "Author Response",
"response": "1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for NOT using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript. Response: Both illumina and PacBio reads have been trimmed only for adaptor removal. No quality trimming was performed. (The manuscript has been corrected): “Quality-filtered reads from the Illumina” has been replaced by “Trimmed reads from the Illumina” “These data was trimmed of adapters and low-quality sequences,…” has been replaced by “These data was trimmed of adapters,…” “Prior to assembly, Illumina and PacBio raw reads were trimmed for quality and adaptor removal using bbduk.sh” has been replaced by “Prior to assembly, Illumina raw reads and PacBio CCS reads were trimmed for adaptor removal using bbduk.sh” Minor comments/suggestions: 1. For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence (CCS) reads?Response: CCS PacBio reads were processed within MaSuRCA software 2. The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ? Response: DNA extracted from the lyophilized leaves was divided into HMW and LMW fractions, and each was used separately to make PacBio libraries. The best PacBio SMRT cell yielded 4G of data on 163,000 spots, with an average read size of 24,468 bases. 3. The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and an estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 = 236X. Please correct or explain further. Response: We agree with the reviewer. The manuscript have been corrected:“These data was trimmed of adapters and low-quality sequences, yielding a clean set of 936,053,040 reads, representing 160× genome coverage,” has now been replaced by “These data was trimmed of adapters and low-quality sequences, yielding a clean set of 936,053,040 reads, representing 236x genome coverage,” 4. In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided. Response: Based on the 21-mer distribution analysis, the duplicated fraction of the genome is estimated to be 3,19%. The revised version of the manuscript has been corrected: “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % (Figure 2).” Has been replaced by “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % and the duplicated fraction of the genome is estimated to be 3,19% (Figure 2).” 5. For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined. Response: We agree. The lineage parameter used is “embryophyta_odb9”. (The manuscript has been corrected): “To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with Arabidopsis lineage-specific orthologous groups31,” has been replaced by “To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with “embryophyta_odb9” lineage-specific orthologous groups31,” 6. The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries. Response: We agree with the reviewer. The work is in progress and we plant to publish the organellar genomes soon in a separate manuscript."
},
{
"c_id": "5438",
"date": "04 May 2020",
"name": "Rachid Mentag",
"role": "Author Response",
"response": "Answers to Reviewer #1 Major reservation: 1. Both Illumina as well as PacBio reads have been processed and trimmed before being used as an input to the MaSuRCA software. However, the instructions for the MaSuRCA software explicitly ask for NOT using any third-party tools, or performing any trimming, cleaning or error correction on Illumina reads, as it will most likely lead to a deteriorated assembly (See https://github.com/alekseyzimin/masurca#overview). It is therefore strongly suggested that the authors try an assembly with the raw Illumina reads directly, as it might lead to a better assembly. If such an analysis was already performed but the assembly was worse than the current version, please provide this information in the manuscript. Answer.We agree with the reviewer. Both illumina and PacBio reads have been trimmed only for adaptor removal. No quality trimming was performed. The manuscript has been corrected: “Quality-filtered reads from the Illumina” has been replaced by “Trimmed reads from the Illumina” “These data was trimmed of adapters and low-quality sequences,…” has been replaced by “These data was trimmed of adapters,…” “Prior to assembly, Illumina and PacBio raw reads were trimmed for quality and adaptor removal using bbduk.sh” has beenreplaced by “Prior to assembly, Illumina raw reads and PacBio CCS reads were trimmed for adaptor removal using bbduk.sh” Minor comment/suggestion: 1- For PacBio data, please specify the form of processed reads that were input to the MaSuRCA software: were they the raw PacBio reads, or the Circular Consensus Sequence (CCS) reads? Answer. CCS PacBio reads were processed within MaSuRCA software. The manuscript has been corrected: “Prior to assembly, Short and long reads were…” has been replaced by “Prior to assembly, Illumina raw reads and PacBio CCS reads were …” 2- The authors report that the genomic DNA was size-selected for fragments above 2kb. What was the approximate maximum size of input DNA for PacBio library preparation. Also, what was the maximum size of PacBio reads (e.g after the CCS step) ? Answer.DNA extracted from the lyophilized leaves was divided into HMW and LMW fractions, and each was used separately to make PacBio libraries. The best PacBio SMRT cell yielded 4G of data on 163,000 spots, with an average read size of 24,468 bases. 3- The raw coverage obtained from Illumina data is reported as 160X. But as per my calculations, based on number of trimmed bases in table 1 and a estimated genome size of 573 Mb, I obtain 135,539,587,270 / 573,000,000 = 236X. Please correct or explain further. Answer.We agree with the reviewer. The manuscript have been corrected: “….yielding a clean set of 936,053,040 reads, representing 160× genome coverage,” has now been replaced by “…yielding a clean set of 936,053,040 reads, representing 236x genome coverage,” 4- In addition to the observed heterozygosity, the repeat content of the genome could be an important reason for a fragmented assembly. The k-mer analysis can also be used to estimate the % of genome that is comprised of repeats. It will be great if this estimate is provided. Answer.Based on the 21-mer distribution analysis, the duplicated fraction of the genome is estimated to be 3,19%. The revised version of the manuscript has been corrected: “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % (Figure 2).” Has been replaced by “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58 % and the duplicated fraction of the genome is estimated to be 3,19% (Figure 2).” 5- For the BUSCO analysis, the authors report using the Arabidopsis lineage-specific orthologous groups. However, from the BUSCO manual, the only valid value for the lineage parameter seems to be “embryophyta_odb9”, which internally uses Arabidopsis hmms for Augustus-based gene predictions, but the member proteins are form multiple species. If this is the lineage parameter the authors used, please specifiy it as “embryophyta_odb9” rather than Arabidopsis, or provide more details on how the Arabidopsis BUSCO groups were determined. Answer. We agree with the reviewer. The lineage parameter used was “embryophyta_odb9”. The manuscript has been corrected: “To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with Arabidopsis lineage-specific orthologous groups...” has been replaced by “To assess the completeness of the final assembly, a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 software approach was used with “embryophyta_odb9” lineage-specific orthologous groups...” 6- The genome sequences of the organelles mitochondria and chloroplast are also very important in investigating the biology of any species, and also serve as important resource for evolutionary and phylogenetic studies. Therefore, it will be great if instead of completely removing the organellar genomes, the authors provide them as separate set of sequences, in separate files and ideally as separate GenBank entries. Answer.We agree with the reviewer. The work is in progress and we plan to publish the organellar genomes soon in separate manuscripts."
}
]
},
{
"id": "38276",
"date": "18 Sep 2018",
"name": "Granger Sutton",
"expertise": [
"Reviewer Expertise Whole genome shotgun assembly",
"pan-genome analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article “First draft genome assembly of the Argane tree (Argania spinosa)”, the authors present the draft assembly of the Argane tree using a combination of Illumina and PacBio reads. An argument for the importance of the Argane tree to the agriculture and economy of the endemic region is given. The assertion that “the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes” seems reasonable based on the use of previous genome sequences for important agricultural resources. This article does not claim much beyond providing a reasonable draft genome for A. spinosa. The primary evidence for reasonable quality is three-fold: a k-mer analysis of genome size using Jellyfish, 94% of Illumina reads mapping back to the assembly, and a BUSCO analysis showing 89% full length and 4.3% partial length gene matches for these single copy genes. This is probably sufficient to claim reasonable quality and the usefulness of the genome for downstream research given the contiguity shown by the N50 contig size.\nThe authors do miss an opportunity to look more closely at the haplotype separation that may be occurring as they speculate possibly leading to a somewhat larger genome size than indicated by Jellyfish. I’m not sure why the number of BUSCO genes with more than one full or partial length match was not given – perhaps there were none? The implication that heterozygosity is uniform “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58%” seems unfounded without more evidence nor is it clear that is the model resulting in this estimate.\nLastly the grammar would benefit from editing. For example, “A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing.” Might be better as “A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5439",
"date": "04 May 2020",
"name": "Rachid Mentag",
"role": "Author Response",
"response": "Answers to Reviewer #2 1- The authors do miss an opportunity to look more closely at the haplotype separation that may be occurring as they speculate possibly leading to a somewhat larger genome size than indicated by Jellyfish. I’m not sure why the number of BUSCO genes with more than one full or partial length match was not given – perhaps there were none? The implication that heterozygosity is uniform “The double peak of k-mer distribution indicates heterozygosity whose rate is estimated to be 1.58%” seems unfounded without more evidence nor is it clear that is the model resulting in this estimate. Answer. We agree with the reviewer. The manuscript has been corrected: “..which showed that the assembly contained 89% (1271 genes) of complete and 4.3% (62 genes) of partial sequences that were Arabidopsis orthologs.” has been replaced by “. Thus, from a total of 1,440 BUSCO genes, 1291 genes (89%) were complete (1179 in single copy and 112 duplicated), 62 genes (4.3%) were represented partially while 87 genes (6%) were missing” from the assembly. 2- Lastly the grammar would benefit from editing. For example, “A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing.” Might be better as “A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”. Answer. We agree with the reviewer. The manuscript has been corrected: \" A reliable annotation is highly dependent on transcriptomic research, and sequencing of Argane transcriptome analysis of different parts and developmental stages or the plant is ongoing\" has been replaced by \" A reliable annotation is highly dependent on transcriptomic data, analysis and research. Sequencing of different parts and developmental stages of the Argane transcriptome is ongoing.”"
}
]
}
] | 1
|
https://f1000research.com/articles/7-1310
|
https://f1000research.com/articles/9-319/v1
|
01 May 20
|
{
"type": "Review",
"title": "Energy metabolism and sources of oxidative stress in wooden breast - a review",
"authors": [
"Shawna M. Hubert",
"Giridhar Athrey",
"Shawna M. Hubert"
],
"abstract": "Modern broilers show dramatic growth over a short interval and contribute directly to the success of the poultry meat industry. The growth performance of commercial broilers is a result of genetic selection for \"performance traits\", such as body size, meat yield, and feed conversion rate. However, due to the rapid growth rate of modern commercial broilers, several growth-related conditions have arisen, increasing economic losses and consumer concerns. Among the most economically consequential is the muscle disorder called wooden breast. Together with associated myopathies such as white striping and spaghetti meat, wooden breast is causing losses of $200 million a year in the U.S. alone and occurs worldwide. No causative factors are known for wooden breast to date. Wooden breast can affect over 80% of broilers in a flock, yet no methods of amelioration are currently available. Overall, the evidence suggests that wooden breast is a genetic, age-dependent condition associated with fast growth rate. The primary features of wooden breast are muscle degeneration and fibrosis, high levels of oxidative stress, hypoxia, and altered energy metabolism. Recent work has also implicated reduced pectoral vessel density in the pathogenesis of wooden breast. This review examines the history of myopathies in commercial broilers and the relationship of myopathies to metabolism and oxidative performance. This review summarizes the foundational knowledge of wooden breast and provides a platform for further investigation of wooden breast.",
"keywords": [
"wooden breast",
"broiler chicken",
"production efficiency",
"myopathy",
"meat quality"
],
"content": "Introduction\n\nThroughout the last 60 years the poultry industry has worked to increase growth rate, muscle development, and maturation of chickens in order to maximize production efficiency and keep poultry meat prices low (Bohren, 1953; Chambers et al., 1981; Elwinger et al., 2016; Fairfull & Chambers, 1984; Jaap, 1963; Merritt et al., 1962; Paxton et al., 2010; Remignon et al., 1994; Skoglund et al., 1966; Tallentire et al., 2018). These improvements have been accomplished through selective breeding and carefully maintaining multiple generations of broiler breeder flocks. However, through these efforts, the genetic diversity of current commercial flocks has become limited, allowing for numerous health concerns to arise (Julian 2005; Paxton et al., 2010; Tallentire et al., 2018). These concerns include weak immunity, vascular insufficiency, skeletal impairment, heat intolerance and a class of disorders termed myopathies which are generally considered meat quality issues rather than bird health concerns (Chen et al., 2019; Clark & Velleman, 2016; Cruz et al., 2017; Hubert et al., 2018; Kuttappan et al., 2012; Kuttappan et al., 2017a; Kuttappan et al., 2017b; MacRae et al., 2006; Malan et al., 2003; Mazzoni et al., 2015; Scheele, 1997; Siller, 1985; Sihvo et al., 2014; Tarrant et al., 2017; Tallentire et al., 2018; Tijare et al., 2016; Trocino et al., 2015; Velleman et al., 2018; Wideman et al., 2013).\n\nGenerally, a myopathy constitutes a disorder of the skeletal muscle, often localized to a specific muscle tissue, in which impaired cell structure and metabolism generate macroscopic symptoms and muscle dysfunction. Myopathies can occur due to inheritance, chronic immune and endocrine disruptions, and environmental stimuli. For example, wing-flapping induces deep pectoral myopathy (DPM) (Siller, 1985). Additionally, antibiotics such as monensin (Umemura et al., 1984), hormone dysregulation such as the inhibition of prostaglandin (McLennan, 1985) and degeneration of mitochondria due to feed ingredients such as Senna seeds (Cavaliere et al., 1997) have been observed to cause myopathies in poultry. Inherited (Asmundson & Julian, 1956; Asmundson et al., 1966) and nutritional muscular dystrophy (Weinstock et al., 1955) were the first myopathies to be investigated and are still arising in poultry over 60 years later. Due to the different modalities of inducing myopathies, it is difficult to pinpoint the pathophysiology of myopathies. However, myopathies have become a prevalent consumer issue and economic concern in today's fast growth commercial broiler lines, necessitating intense investigation. Recent literature has focused on the three most commonly observed broiler breast myopathies, DPM, white striping (WS) and wooden breast (WB), and a newly observed myopathy termed “spaghetti meat” (SM).\n\nAlthough they are all classified as myopathies of the breast, DPM impacts the pectoralis minor while WS, WB, and SM impact the pectoralis major. DPM was first observed in broilers in 1980 (Richardson et al., 1980) and is commonly referred to as green muscle disease due to a characteristic green coloring of the pectoralis minor in affected birds. Since then, numerous investigators have concluded that the massive pectoralis major of commercial broiler suffocates the pectoralis minor, resulting in hemorrhage and necrosis of the muscle (Bailey et al., 2015; Grunder et al., 1984; Kuttappan et al., 2016; Lien et al., 2012; Siller, 1985; Velleman, 2015; Wight & Siller, 1980). The green coloring is caused by the release of iron from the hemorrhaged erythrocytes as they break down. Histological characterization of DPM by Wight & Siller (1980) showed anucleated myofibers surrounded by fibrous and adipose tissues, and disintegration of the sarcoplasmic reticulum, mitochondria, and Z-lines. These studies suggest that DPM is a result of artificial selection for high growth rate and increased breast yield.\n\nWS is so named based on the appearance of the affected pectoralis major, marked by white striations that run parallel to the muscle fibers. Recent studies have determined that the manifestation of white striping is not only altered appearance but also a decrease in mineral and protein content, water holding capacity, and an increase in fat and collagen content (Bailey et al., 2015; Kuttappan et al., 2013b; Kuttappan et al., 2013c; Kuttappan et al., 2012; Kuttappan, 2012; Kuttappan et al., 2013a; Mazzoni et al., 2015; Mudalal et al., 2015; Owens, 2014; Petracci et al., 2013; Petracci et al., 2014; Sihvo et al., 2014; Trocino et al., 2015; Vignale et al., 2017). At the histological level, variable fiber size, necrosis, loss of cross striations, multinucleated cells, interstitial inflammation and fibrosis, lipidosis, and mononuclear cell infiltration are seen (Kuttappan et al., 2013c; Kuttappan et al., 2013a; Mazzoni et al., 2015; Trocino et al., 2015). Few studies have investigated the molecular mechanisms of WS. Vignale et al. (2017) focused on elucidating the activity of genes related to protein synthesis and degradation, such as IGF-1, atrogin-1, insulin receptor, and MuRF1. Their results indicated that MuRF1 and atrogin-1 are significantly upregulated in WS birds, while IGF-1 was significantly down-regulated. Vignale et al. (2017) suggested that these changes in gene expression indicated that fast growth commercial broilers have a higher rate of muscle degradation, which leads to the development of WS. Furthermore, Pampouille et al. (2018) utilized a genome-wide association study of quantitative trait loci, which resulted in the identification of possible genetic and molecular markers for WS and provided evidence for polygenic inheritance of the condition. These include several genes involved in muscle structure and metabolism, some known to be involved in neuromuscular disorders (Pampouille et al., 2018).\n\nOne of the recently identified myopathies known as WB is a significant concern for the poultry industry due to its worldwide occurrence high incidence (Bailey et al., 2015; Clark & Velleman, 2016; Cruz et al., 2017; Mutryn et al., 2015; Petracci et al., 2019; Sihvo et al., 2017). WB is characterized by an abnormally hard or “wooden” breast, with hardness originating at the cranial region in the least severe cases and extending the full length of the breast, from the cranial to the caudal region in the most severe cases. Breasts affected by WB often appear to have a medial ridge extending from the cranial to caudal region and meat quality factors such as texture, shear force, pH and water holding capacity are all impacted (Bailey et al., 2015; Clark & Velleman, 2016; Coble et al., 2014; Cruz et al., 2017; Kuttappan et al., 2016; Kuttappan et al., 2017b; Mazzoni et al., 2015; Sihvo et al., 2014; Sihvo et al., 2017; Soglia et al., 2016; Tijare et al., 2016; Trocino et al., 2015; Velleman & Clark, 2015; Velleman et al., 2018).\n\nSM, the most recently described myopathy impacting the poultry industry, also primarily affects the pectoralis major. It manifests as impaired muscle structure, resulting in the separation of muscle fiber bundles, and generally impacts the cranial portion of the breast fillet (Baldi et al., 2018). WS and SM co-occur and share histological features such as increased perimysial and endomysial degradation of connective tissues, infiltration of inflammatory cells, and thin and split fibers surrounded by loose connective tissue (Baldi et al., 2018). Collagen of SM affected breasts is immature and has fewer cross-links compared to normal breast fillets (Baldi et al., 2019). Furthermore, SM is higher in moisture content, but lower in protein and fat content than WS affected breasts (Baldi et al., 2018; Baldi et al., 2019). SM affected breasts are downgraded for quality and used in further processed products, resulting in economic losses (Baldi et al., 2018; Baldi et al., 2019). Nutritional interventions for SM through altered arginine:lysine ratios were investigated by Zampiga et al. (2019), resulting in significantly reduced incidence at the highest arginine:lysine ratio. This reduction in SM incidence was hypothesized to be due to enhanced vasodilation and better blood flow to the muscle through increased production of nitric oxide via the arginine-nitric oxide pathway (Zampiga et al., 2019). However, the authors noted that the reduction in lysine could also be responsible for the decreased incidence of SM, as other researchers have shown increases in breast muscle myopathies due to increased dietary lysine concentrations (Cruz et al., 2017; Zampiga et al., 2019).\n\nChicken breast is the most consumed meat in the U.S., and the quality and safety of this food commodity are significant both from an economic and food security standpoint. Broiler breast myopathies decrease meat quality by altering the muscle ultrastructure, which in turn impacts texture, tenderness, and in some cases, flavor. With high incidence rates, the annual economic losses due to broiler breast myopathies in the U.S. are roughly $200 million and increasing (Kuttappan et al., 2016). The pectoralis major is known as the breast when discussed as a meat product and is the most purchased cut, while the pectoralis minor is known as the tender. Chicken breast meat is consumed widely due to the high protein content and low-fat content. However, due to these myopathies, this desirable protein and fat ratio is altered. WB affected meat, for instance, has higher fat content and lower protein content (Kuttappan et al., 2017a; Mazzoni et al., 2015; Mudalal et al., 2015; Owens, 2014; Petracci et al., 2014; Soglia et al., 2016; Tasoniero et al., 2017; Tijare et al., 2016). The percentage of fat increase in breast meat affected by WB is 1.2-1.3% (Soglia et al., 2016; Tasoniero et al., 2016).\n\nFurthermore, as consumers have become aware of these myopathies, there is an increasing rejection of WB affected meat, as well as discussion on social media (Burginger, 2019; Crews, 2017; Elder, 2017; Gee, 2016; Johnson, 2018; Jones, 2016; Keiger, 2017; Ngo, 2017; Pellegrini, 2017; Petreycik, 2019; Picchi, 2016; Prescott, 2017; Rainey, 2016; Versace, 2019; Walansky, 2017). Often, the public is misinformed on the source of these undesirable characteristics and misattribute them to questionable production practices or confuse them with genetically modified organisms. Although such views are incorrect, they contribute to misinformation about animal agriculture and exacerbate food waste. Thus, it is imperative to improve education for the public on these crucial issues while solutions are found and implemented.\n\n\nLiterature review\n\nAlthough selective breeding of production animals developed around specific phenotypes, scientific advances in genetics and nutrition have created the ability to maximize the efficiency of selection using genotype data. However, trait heritability has been the basis of selection since roughly the 1980s (Chambers et al., 1981; Fairfull & Chambers, 1984), and the implementation of genetic and genomic data in poultry selection is still in its infancy. Le Bihan-Duval et al. (2008) reported that growth and body composition traits such as body weight and abdominal fat were heritable (h2 = 0.49 and 0.48 respectively), as were muscle characteristics such as glycolytic potential and muscle fiber cross-sectional area (h2 = 0.43 and 0.41 respectively) in broilers. This study also reports that glycolytic potential was negatively correlated to meat quality traits such as color, drip loss and shear force, and overall breast muscle weight (Le Bihan-Duval et al., 2008).\n\nChicken breast muscle physiology is well studied as it is the most valuable portion of the carcass and has been for more than 30 years. In 1994, Remignon et al. evaluated the muscle characteristics of slow-growth and fast-growth chicken lines to isolate muscle characteristics specific to growth-rate. Their study found no differences in muscle fiber type at 55 weeks (Remignon et al., 1994). The breast was composed only of Type IIB fast-twitch fibers, and cross-sectional areas were larger and more numerous in the fast-growth lines compared to the slow growth line. However, some studies have identified a small percentage (0.5-10%) of Type IIA fibers in slow-growth and laying type chickens (Branciari et al., 2009; Clark & Velleman, 2016; Dransfield & Sosnicki, 1999; MacRae et al., 2006; Remignon et al., 1995; Scheuermann et al., 2004; Velleman et al., 2018; Velleman, 2007).\n\nThe characteristics of the chicken breast muscle are primarily responsible for its popularity with consumers. It is high in protein and low in fat, while typically low in cost. Its stereotypical \"taste of chicken\" allows it to pair well with most recipes and it is functional as a comminuted product, allowing it to have a variety of further processed food products, and is amenable to easy preparation. These desirable attributes are based not only on its Type IIB fibers and low-fat content but also the size and density of fibers, post-mortem pH, and water holding capacity (Baldi et al., 2018; Berri et al., 2007; Dransfield & Sosnicki, 1999; Fanatico et al., 2007; Golzar Adabi & Demirok Soncu, 2019; Le Bihan-Duval et al., 2008; Lonergan et al., 2003; Maxwell et al., 2018; Mazzoni et al., 2015; Mueller et al., 2018; Papa & Lyon, 1989; Petracci & Cavani, 2012; Petracci et al., 2015; Rosser & George, 1986; Scheuermann et al., 2003; Smith & Fletcher, 1988; Trocino et al., 2015).\n\nSpecifically, water holding capacity (WHC) impacts the flavor and toughness of the breast through the amount of water retained or lost during cooking, as well as the ability of the meat to absorb marinades and flavorings. WHC is directly related to the post-mortem pH, with a low pH resulting in reduced WHC and a high pH resulting in increased WHC. However, pH only accounts for about one-third of WHC, with the remainder due to the steric effects of muscle proteins. During rigor, the myofibrillar proteins form irreversible bonds, and muscle contraction occurs, reducing space for water storage. As rigor resolves, the contraction reduces, and several other muscle proteins such as z-lines have degraded, allowing the charged actomyosin complex to recruit water molecules and thus contributing to WHC. The specific combination of fat, protein, pH, and WHC is mainly responsible for the competence of chicken in comminuted products such as chicken nuggets. Deviations of these attributes result in downgrades in product quality, reduced consumer acceptance, and economic losses (Betti et al., 2009; Kato et al., 2018; Kuttappan, 2012; Pellegrini, 2017; Petracci et al., 2019).\n\nFirst identified roughly 15 years ago and intensely investigated during the last five years, many etiologies for WB have been proposed. The most commonly investigated etiologies include nutritional deficiencies and toxicities, exercise induction, and hypoxia of the pectoralis major (Bailey et al., 2015; Bodle et al., 2018; Chen et al., 2019; Clark & Velleman, 2016; Cruz et al., 2017; Guetchom et al., 2012 Lilburn et al., 2019; Livingston et al., 2019a; Meloche et al., 2018; Papah et al., 2017; Soglia et al., 2016; Sihvo et al., 2018; Trocino et al., 2015; Velleman & Clark, 2015; Velleman et al., 2018). Despite this intense study, the molecular mechanisms and associated disease pathways driving WB remain unknown. There is, however, a consensus that WB is associated with the selection for fast growth-rate, occurring at highest frequency in the largest birds; slow growth varieties are not impacted (Hubert et al., 2018; Kong et al., 2017; Meloche et al., 2018; Mudalal et al., 2015; Mutryn et al., 2015; Velleman & Clark 2015). Although similarities exist between WB and well-known myopathies such as WS, the histological characteristics of WB are well described. They include muscle fiber damage, interstitial fibrosis, infiltration of macrophages and an increase in fat and collagen content (Bowker et al., 2019; Clark & Velleman, 2016; Lilburn et al., 2019; Mazzoni et al., 2015; Meloche et al., 2018; Sihvo et al., 2017; Soglia et al., 2016; Sihvo et al., 2014; Sihvo et al., 2018; Velleman et al., 2018; Velleman & Clark, 2015).\n\nAt the molecular level, gene expression and metabolomics studies have shown that oxidative stress is one of the main features of WB tissue (Abasht et al., 2016; Clark & Velleman, 2016; Hubert et al., 2018; Kong et al., 2017; Mutryn et al., 2015; Papah et al., 2018; Sihvo et al., 2017). Reactive oxygen species (ROS), the free radicals produced in vivo by specialized enzymes such as NADPH-oxidase, nitric oxide synthase and myeloperoxidase, mitochondrial respiration and the monooxygenase activity of cytochrome p450, act as modulators of gene expression, increased cell proliferation, prevention of cell division, apoptosis, necrosis and cell death, and are responsible for the damage occurring (Weidinger & Kozlov, 2015) in oxidative stress (Bonnard et al., 2008; Halliwell, 2007; Mutryn et al., 2015; Netzer et al., 2015). In healthy tissues, ROS production is combated by antioxidants, preventing cellular damage. However, in tissues under oxidative stress, the ratio of ROS to antioxidants is highly imbalanced, and long term cellular damage can occur. An example of this damage is direct modulation of the sodium/potassium-ATPase channels, resulting in changes in ion balance, which trigger changes in cellular calcium metabolism, causing increased concentrations of intracellular free calcium and subsequent cellular impairment (Sims & Muyderman, 2010). This characterization provides tremendous insight into the physiological occurrences of WB and provides an excellent foundation for the development of further investigations.\n\nThe poultry industry has made immense progress at increasing growth rate, breast yield, and feed efficiency of commercial broilers through careful selective breeding regimens and maintenance of pedigree flocks (Clark & Velleman, 2016; Collins et al., 2014; Dransfield & Sosnicki, 1999; Fanatico et al., 2007; MacRae et al., 2007; MacRae et al., 2006; Mazzoni et al., 2015; Petracci & Cavani, 2012; Siller, 1985; Velleman & Clark, 2015). However, the quality of the meat produced is directly related to the morphological structure of the muscle, and growth-related selection has changed this structure. Intense selection has resulted in decreased capillary blood supply to the pectoralis major and minor, reduced connective tissue spacing between myofibers and muscle fiber bundles, and increased degeneration of myofibers (Baldi et al., 2018; Berri et al., 2007; Dransfield & Sosnicki, 1999; Fanatico et al., 2007; Kuttappan et al., 2012; Lilburn et al., 2019; MacRae et al., 2007; MacRae et al., 2006; Mahon, 1999; Mazzoni et al., 2015; Mitchell, 1999; Petracci & Cavani, 2012; Scheele, 1997; Sihvo et al., 2014; Sihvo et al., 2018; Soglia et al., 2019; Trocino et al., 2015). Furthermore, myofibers of fast-growth commercial birds are three to five times larger than those of slower-growing birds (Dransfield & Sosnicki, 1999; Velleman, 2015).\n\nThese muscular changes associated with selection for rapid growth in early life are exacerbated in WB (Clark & Velleman, 2016; MacRae et al., 2007; MacRae et al., 2006; Mazzoni et al., 2015; Petracci & Cavani, 2012; Soglia et al., 2016; Trocino et al., 2015; Velleman & Clark, 2015; Velleman et al., 2018). Some researchers hypothesized as early as 1999 (Mahon, 1999) that poultry growth rates might be hitting a maximum threshold at which myofiber metabolism would be compromised by its increased size and the inability of oxygen, nutrients, and waste to diffuse across the fiber. This idea is supported through the demonstration of increased numbers of necrotic, basophilic, and hyaline fibers, and fibers with NADH rich rims or negative cores. These features are indicative of mitochondrial dysfunction and altered oxidative metabolism in fast growth rate commercial broilers when compared to laying hens (MacRae et al., 2007; MacRae et al., 2006). Multiple recent histological studies of WB have demonstrated that the same lesions are observed in both affected and unaffected birds, but affected birds show significantly more lesions (Clark & Velleman, 2016; Mazzoni et al., 2015; Sihvo et al., 2017; Sihvo et al., 2018; Trocino et al., 2015; Velleman et al., 2018; Velleman & Clark, 2015). Three of these investigations have surveyed and found an absence of the histological characteristics of WB in the slow-growth broiler or layer lines (Clark & Velleman, 2016; Velleman & Clark, 2015; Velleman et al., 2018).\n\nOthers have investigated the pathophysiology of WB using transcriptomics, metabolomics, and proteomics approaches. When investigating affected and unaffected birds of the same lines, these studies have identified pathways showing differential regulation to include those associated with cellular movement, proliferation, assembly, function and maintenance, protein synthesis, post-translational modification, protein folding and carbohydrate metabolism (Abasht et al., 2016; Cai et al., 2018; Hubert et al., 2018; Kong et al., 2017; Kuttappan et al., 2017b; Kuttappan et al., 2017a; Mutryn et al., 2015; Papah et al., 2018; Schilling et al., 2017). When comparing both affected and unaffected fast-growth commercial broilers against slow-growth broilers, they have observed differential regulation of the same pathways as discussed above. However, they also observed differential regulation of disease pathways involved in organismal injury or abnormalities and abnormal development and morphology of muscle (Hubert et al., 2018; Kong et al., 2017; Velleman & Clark, 2015). Additionally, comparison of fast-growth and slow-growth varieties indicated that mitochondrial activities such as oxidative phosphorylation and the tricarboxylic acid cycle are downregulated in breast muscles of fast-growth broilers (Kong et al., 2017). These observations support the histological characteristics of WB. Furthermore, the molecular signatures of WB are suggestive of several diseases and disorders. The most significant included gastrointestinal disease, cardiovascular disease, hepatic system disease, neurological disease, and cancer (Abasht et al., 2016; Hubert et al., 2018; Kong et al., 2017; Kuttappan et al., 2017a; Mutryn et al., 2015).\n\nAlthough differences in gene expression and the subsequent alterations to physiological pathways can be detected when comparing unaffected and affected WB samples, the resolution is weak and cannot provide a definitive pathology. Unaffected samples may not have developed the condition at the time of collection or are asymptomatic (Abasht et al., 2016; Hubert et al., 2018; Kuttappan et al., 2017a; Kuttappan et al., 2017b; Sihvo et al., 2017). The utilization of slow-growth varieties as a negative control for WB investigations can provide a clearer representation of the associated changes. Additionally, such a comparison would increase our understanding of genes and pathways that are concurrently important for WB and production traits such as growth rate and feed efficiency. Data from these investigations suggest that unaffected WB tissue may not be an adequate negative control for the determination of the molecular characteristics and pathophysiology of WB.\n\nThe mitochondrion has crucial roles in cellular respiration, as well as several other factors specific to WB. As determined by Mutryn et al. (2015), gene expression studies of WB affected muscle samples have indicated intracellular calcium accumulation that has the potential to impair cell membrane integrity. Interestingly, the mitochondria regulate cytosolic calcium concentrations, which in turn regulate the cellular reduction-oxidation reactions of a variety of transcription factors and cellular enzymatic reactions, control cellular, and mitochondrial metabolic pathways, and manage mitochondrial ROS production (de Oliveira et al., 2017; Wallace, 2013). Furthermore, calcium overload has the potential to activate proteases and lipases within the cell, resulting in myofiber degeneration, a commonly observed histological feature of WB tissue (Halliwell, 2007; Mutryn et al., 2015). To this end, the role of mitochondria in WB occurrence or severity remains unclear. This gap in knowledge is surprising due to the mitochondrion's significance for muscle energetics, cell signaling, and death, which are all known features in WB progression.\n\nIt is well known that mitochondria are commonly called the powerhouse of the cell and that they function in oxidative metabolism to provide energy in the form of ATP to the body as well as facilitate the removal of wastes and act in cell signaling, differentiation and programmed cell death (Angelini et al., 2009; Bonnard et al., 2008; de Oliveira et al., 2017; Irwin et al., 2008; Koch, 2016; Latorre-Pellicer et al., 2016; Liao et al., 2015; Muir et al., 2016; Moreno-Loshuertos et al., 2011; Netzer et al., 2015; Sims & Muyderman, 2010; Tuppen et al., 2010; Velarde, 2013; Wallace & Chalkia, 2013). However, the mitochondrion is the only organelle in the cell with a genome (Koch, 2016; Latorre-Pellicer et al., 2016; Tuppen et al., 2010; Wallace & Chalkia, 2013; Yarham et al., 2010). Mitochondrial DNA (mtDNA) is small, circular, and clonally inherited from the mother. It consists of two regions; the non-coding region, which controls mtDNA, and the coding region, which codes for tRNAs, rRNAs, and 13 cellular energy production genes, and contains no introns (Latorre-Pellicer et al., 2016; Moreno-Loshuertos et al., 2011; Muir et al., 2016; Takemoto et al., 1999; Tuppen et al., 2010; Wai et al., 2008; Wallace & Chalkia, 2013). Mitochondrial DNA is haploid and does not recombine; it is typically stable over time, but due to its few repair mechanisms, it tends to have a much higher mutation rate than nuclear DNA, and mutations are commonly deleterious.\n\nThe number of mitochondria in a cell varies significantly by type and tissue, and they often have more than one genome sequence, a common condition known as heteroplasmy (Abbott et al., 2014; Carelli et al., 2015; DiMauro & Schon, 2001; Lehmann et al., 2015; Luo et al., 2018; Payne et al., 2013; Tuppen et al., 2010; Wallace & Chalkia, 2013; Wai et al., 2008). This heteroplasmy often acts as a sort of shield for an individual carrying mutant mtDNA mutations. Over a lifetime, the percentage of mutant mtDNAs increases, resulting in a decline in the individuals bioenergetic capacity below the minimum threshold and symptoms of the pathogenic mtDNA mutation ensue (Abbott et al., 2014; Carelli et al., 2015; Lehmann et al., 2015; Latorre-Pellicer et al., 2016; Wallace & Chalkia, 2013). Due to this unique nature, pathogenic mtDNA mutations and the conditions caused by them are widespread, continually arising, can be localized to a specific tissue, are familial, and often age-related (Lehmann et al., 2015; Moreno-Loshuertos et al., 2011; Tuppen et al., 2010; Wai et al., 2008; Weber et al., 1997; Zeviani et al., 1991). This description fits the developmental stages observed in WB, as it arises at different ages, affects a high percentage, and occurs in varying severities throughout a broiler flock.\n\nA critical trait of WB posing a dilemma for determining its pathology is its apparent localization to the breast muscle only. However, diseases caused by mitochondrial mutations typically localize to a tissue or region of the body, and the observation of both histological and molecular markers of oxidative stress and myocyte degeneration reveals their role in the pathogenesis of WB. We can determine the role of mitochondrial activity in WB through the use of next generation sequencing technologies and bioinformatics. However, investigations into human diseases in which mitochondria have recently been implicated such as diabetes, obesity, metabolic syndrome, stroke, Alzheimer's, and cancer have demonstrated that an understanding of the molecular characteristics of the disease is instrumental in determining the involvement of mitochondrial-related bioenergetic alterations (Angelini et al., 2009; Barja & Herrero, 2000; Bonnard et al., 2008; D’Souza et al., 2011; Glancy et al., 2017; Latorre-Pellicer et al., 2016; Long et al., 2012; Maurya et al., 2018; Moreno-Loshuertos et al., 2011; Muir et al., 2016; Sims & Muyderman, 2010; Velarde, 2013; Wallace & Chalkia, 2013; Yarham et al., 2010). It is through these mechanisms that mitochondrial mutations can have significant physiological impacts; therefore, it is necessary to characterize the mitochondrial structure and gene expression related to WB.\n\nIn contrast, based on the evidence for oxidative stress and hypoxia in WB, some hypotheses have suggested a genomic and nutritional interaction based on the high energy, soy-based diet typical of the commercial broiler (Cruz et al., 2017; Haug et al., 2007; Koppenol et al., 2015; Li et al., 2019; Livingston et al., 2019b). Although many nutritional interventions were assessed, none have managed to decrease the incidence of WB (Bodle et al., 2018; Cruz et al., 2017; Guetchom et al., 2012; Livingston et al., 2019b; Livingston et al., 2019a; Lilburn et al., 2019; Livingston et al., 2019c; Sobotik et al., 2018; Trocino et al., 2015). Still, other hypotheses have gone further to implicate endocrine disruptors such as phytoestrogens from soybean meal in poultry feed, as a potential driver of genomic/nutritional interaction. Phytoestrogens are naturally occurring, plant-derived, biologically active compounds that both structurally and functionally mimic estrogens when in humans and animals (Bacciottini et al., 2007; Cederroth & Nef, 2009; Patisaul & Jefferson, 2010; Stevenson et al., 2014).\n\nSoybean meal, one of the main ingredients in industrial poultry feed, has one of the highest concentrations of phytoestrogens among plant-based feed sources (Cederroth & Nef, 2009; Gjorgovska et al., 2014; Haug et al., 2007; National Research Council et al., 1994; Patisaul & Jefferson, 2010; Payne et al., 2001; Stein et al., 2008; USDA ARS, 2016). Due to their similarity to estrogen and the ability to bind to both estrogen receptor α and β, phytoestrogens act as endocrine-disrupting compounds (Cederroth & Nef, 2009; Gjorgovska et al., 2014; Patisaul & Jefferson, 2010). As estrogen regulates various vital physiological processes such as lipid and glucose metabolism, bone development, sexual maturation, and reproduction, perturbations of this suite of endocrine functions can potentially alter critical system-wide processes. Therefore, the nature and extent to which phytoestrogens drive occurrence and severity of WB deserves further attention.\n\nThe implications of high dietary concentrations of phytoestrogens are also relevant to mitochondrial cell signaling, and oxidative metabolism (Irwin et al., 2008; Liao et al., 2015; Mauvais-Jarvis, 2011; Sarkar et al., 2015; Sims & Muyderman, 2010; Velarde, 2013). One pathway involves alteration of mitochondrial cell signaling and mitochondrial hormonal regulation due to increased exposure to phytoestrogens. Recent work has shown the role of mitochondrial estrogen receptor-ß and its implications for mitochondrial bioenergetics and tumorigenesis (Liao et al., 2015; Velarde, 2013). Additionally, human studies have determined that oxidative stress in skeletal muscle is often induced by dysregulation of the metabolism of energy fuel substrates such as lipids and glucose. The resulting damage to the mitochondria causes an increase in the buildup of ROS and fuel substrates (Bonnard et al., 2008; de Oliveira et al., 2017; Irwin et al., 2008; Liao et al., 2015; Marco et al., 1961; Netzer et al., 2015; Velarde, 2013). The endocrine-disrupting actions of phytoestrogens are capable of inducing metabolic dysregulation, and this is a possible element in the pathogenesis of WB warranting further investigation.\n\nDue to the many vital physiological activities of estrogens, the regulators of estrogen metabolism have become a popular topic in human and animal health. Currently, the most studied are the ω-3 and ω-6 polyunsaturated fatty acids. They boast a myriad of physiological effects including reduction of cholesterol, reduction of circulating levels of 17β-estradiol, reduced binding of estradiol to the estrogen receptor, stabilization of electrical activity of cardiac myocytes, increased apoptosis of cancerous cells, increased 2-hydroxylation of estradiol, and protection for mitochondria (Behling et al., 2015; Cao et al., 2012; de Oliveira et al., 2017; Dikshit et al., 2015; Gómez Candela et al., 2011; Jeromson et al., 2015; Lord et al., 2002; Marco et al., 1961). These effects provide benefits such as reduced risk of cardiovascular and metabolic diseases, and reduced risk of multiple cancer types (Behling et al., 2015; Betti et al., 2009; Cao et al., 2012; Dikshit et al., 2015; Gómez Candela et al., 2011; Jeromson et al., 2015; Lorente-Cebrián et al., 2015; Lord et al., 2002; O’Keefe et al., 1995; Simopoulos, 2002).\n\nMultiple studies have investigated increasing ω-3 fatty acid concentrations in chicken feed, as an indirect way to increase human ω-3 consumption (Betti et al., 2009; Carragher et al., 2016; Haug et al., 2007; Koppenol et al., 2015; Konieczka et al., 2017; Moghadam et al., 2017; Newkirk & Classen, 2002; O’Keefe et al., 1995; Rymer & Givens, 2005; Ratnayake et al., 1989; Zuidhof et al., 2009). A majority of these studies utilized fish-based additives as the source of ω-3s. The fish-based ω supplement did increase the ω-3 content of the muscle, but also caused a fishy taste and decreased the shelf life of the product (Koppenol et al., 2015; O’Keefe et al., 1995; Ratnayake et al., 1989; Rymer & Givens, 2005). More recent studies have investigated plant-based sources of ω-3s such as flax and canola; however, most of these investigations demonstrated reduced growth rates and final carcass weights (Betti et al., 2009; Carragher et al., 2016; Haug et al., 2007; Konieczka et al., 2017; Newkirk & Classen, 2002; Parveen et al., 2013). Currently, the soy- and corn-rich standard poultry industry diet is very high in ω-6s but very low in ω-3s (Dikshit et al., 2015; Haug et al., 2007; O’Keefe et al., 1995; Rymer & Givens, 2005). For humans, the recommended ratio of ω-6:3 is roughly 5:1. Ratios of 2-3:1 have demonstrated improvements in patients with cardiovascular disease, rheumatoid arthritis, asthma and multiple types of cancers, while ratios of 10:1 have shown adverse effects (Gómez Candela et al., 2011; Jeromson et al., 2015; Rymer & Givens, 2005; Simopoulos, 2010; Simopoulos, 2002; Simopoulos, 2016).\n\nBalancing the ω-6:3 content of industry broiler feed could have a positive impact on several conditions, which are currently causing tremendous losses to the poultry industry, including WB, WS, green muscle disease, and sudden death syndrome (SDS) to name a few. Furthermore, none of these studies have attempted to explain the physiological processes which result in the observed changes due to supplemented ω-3s, and few have considered differences in gene expression. Investigation of these processes utilizing multi-disciplinary approaches will help determine the extent to which nutritional modulation of ω-6:3 fatty acid ratios can influence oxidative stress in the muscle.\n\n\nConclusions\n\nAs detailed previously, the most commonly observed theme in WB is a state of oxidative stress in the pectoralis major; the origins and implications of the offending ROS remain unknown to date. Furthermore, the intracellular calcium buildup and NADH displacement indicate dysfunction of the oxidative metabolism machinery and a severe threat to cellular health. Comparative analysis utilizing chicken from multiple varieties (differing growth profiles) to explore transcriptional and genomic changes due to WB in both nuclear and mtDNA would provide increased depth for understanding the pathogenesis of WB and the impact of artificial selection for commercial traits, as would investigating the effects of omega ratios on the molecular signatures inflammation between fast- and slow-growth varieties. Combining these data with further evaluation of WB through \"omics\" technologies can help determine both the origins and implications of this oxidative stress and its actions in the pathogenesis of WB. Through these observations, a clear view of nutritional, age, and genetic interactions could be developed and generate new solutions for WB.\n\n\nData availability\n\nNo data are associated with this article.",
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PubMed Abstract | Publisher Full Text\n\nTuppen HAL, Blakely EL, Turnbull DM, et al.: Mitochondrial DNA mutations and human disease. Biochim Biophys Acta. 2010; 1797(2): 113–128. PubMed Abstract | Publisher Full Text\n\nUmemura T, Nakamura H, Goryo M, et al.: Histopathology of monensin-tiamulin myopathy in broiler chicks. Avian Pathol. 1984; 13(3): 459–467. PubMed Abstract | Publisher Full Text\n\nUSDA ARS: Soy meal defatted raw. 2016; [Accessed: 29 March 2017]. Reference Source\n\nVelarde MC: Pleiotropic actions of estrogen: a mitochondrial matter. Physiol Genomics. 2013; 45(3): 106–109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVelleman SG: Muscle development in the embryo and hatchling. Poult Sci. 2007; 86(5): 1050–1054. PubMed Abstract | Publisher Full Text\n\nVelleman SG: Relationship of Skeletal Muscle Development and Growth to Breast Muscle Myopathies: A Review. Avian Dis. 2015; 59(4): 525–531. PubMed Abstract | Publisher Full Text\n\nVelleman SG, Clark DL: Histopathologic and Myogenic Gene Expression Changes Associated with Wooden Breast in Broiler Breast Muscles. Avian Dis. 2015; 59(3): 410–418. PubMed Abstract | Publisher Full Text\n\nVelleman SG, Clark DL, Tonniges JR: The Effect of the Wooden Breast Myopathy on Sarcomere Structure and Organization. Avian Dis. 2018; 62(1): 28–35. PubMed Abstract | Publisher Full Text\n\nVersace C: You had me at chicken abnormalities and woody breast meat. Tematica Research. 2019; [Accessed: 11 May 2019]. Reference Source\n\nVignale K, Caldas JV, England JA, et al.: Effect of white striping myopathy on breast muscle (Pectoralis major) protein turnover and gene expression in broilers. Poult Sci. 2017; 96(4): 886–893. PubMed Abstract | Publisher Full Text\n\nWai T, Teoli D, Shoubridge EA: The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat Genet. 2008; 40(12); 1484–1488. PubMed Abstract | Publisher Full Text\n\nWalansky A: The poultry industry’s latest problem is hard to chew. Food and Wine. 2017; [Accessed: 11 May 2019]. Reference Source\n\nWallace DC: Bioenergetics in human evolution and disease: implications for the origins of biological complexity and the missing genetic variation of common diseases. Philos Trans R Soc Lond B Biol Sci. 2013; 368(1622): 20120267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWallace DC, Chalkia D: Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring Harb Perspect Biol. 2013; 5(11): a021220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeber K, Wilson JN, Taylor L, et al.: A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle. Am J Hum Genet. 1997; 60(2): 373–380. PubMed Abstract | Free Full Text\n\nWeidinger A, Kozlov AV: Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction. Biomolecules. 2015; 5(2): 472–484. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeinstock IM, Goldrich AD, Milhorat AT: Enzyme Studies in Muscular Dystrophy. I. Muscle Proteolytic Activity and Vitamin E-Deficiency. Exp Biol M. 1955; 88(2): 257–260. Publisher Full Text\n\nWideman RF, Rhoads DD, Erf GF, et al.: Pulmonary arterial hypertension (ascites syndrome) in broilers: a review. Poult Sci. 2013; 92(1): 64–83. PubMed Abstract | Publisher Full Text\n\nWight PA, Siller WG: Pathology of deep pectoral myopathy of broilers. Vet Pathol. 1980; 17(1): 29–39. PubMed Abstract | Publisher Full Text\n\nYarham JW, Elson JL, Blakely EL, et al.: Mitochondrial tRNA mutations and disease. Wiley Interdiscip Rev RNA. 2010; 1(2): 304–324. PubMed Abstract | Publisher Full Text\n\nZampiga M, Soglia F, Petracci M, et al.: Effect of different arginine-to-lysine ratios in broiler chicken diets on the occurrence of breast myopathies and meat quality attributes. Poult Sci. 2019; 98(6): 2691–2697. PubMed Abstract | Publisher Full Text\n\nZeviani M, Gellera C, Antozzi C, et al.: Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNA(Leu)(UUR). Lancet. 1991; 338(8760): 143–147. PubMed Abstract | Publisher Full Text\n\nZuidhof MJ, Betti M, Korver DR, et al.: Omega-3-enriched broiler meat: 1. Optimization of a production system. Poult Sci. 2009; 88(5): 1108–1120. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "66397",
"date": "29 Jul 2020",
"name": "Casey Owens",
"expertise": [
"Reviewer Expertise poultry meat science/myopathies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review is well written. Addition of review on gene expression work would be beneficial and the last part regarding Omegas is not that strong as there is no work to date that shows any relationship to WB. I would suggest deleting that portion or strengthen it.\n\nPlease use \"Pectoralis major\" (or minor) italicized throughout.\n\nPlease use another term other than \"varieties\" such as \"breeds\" Varieties is not commonly used for poultry (more so for plants).\n\nPlease provide a reference for \"First identified roughly 15 years ago...\".\n\nWB characteristics have also been observed in thigh meat so please reword statement about WB localizing in the breast only (ie, maybe change to primarily in the breast) (\"A critical trait of WB.....).\n\nOmega-6 and Omega-3 review is weak in terms of showing no direct relationship to WB. Tie these together more, condense significantly, or delete.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "67834",
"date": "11 Aug 2020",
"name": "Bruno Ronchi",
"expertise": [
"Reviewer Expertise Animal nutrition and metabolism"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review is lacking in the following aspects:\nThe effects of the disease on meat quality of commercial broiler.\n\nThe effects of some disease control programs (e. g. against coccidiosis).\n\nThe impact on productive efficiency and profitability.\n\nThe protective effects of some antioxidants administered with the diet; biochemical signatures potentially useful for diagnosis.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-319
|
https://f1000research.com/articles/9-218/v1
|
30 Mar 20
|
{
"type": "Review",
"title": "A review of open source ventilators for COVID-19 and future pandemics",
"authors": [
"Joshua M. Pearce"
],
"abstract": "Coronavirus Disease 2019 (COVID-19) threatens to overwhelm our medical infrastructure at the regional level causing spikes in mortality rates because of shortages of critical equipment, like ventilators. Fortunately, with the recent development and widespread deployment of small-scale manufacturing technologies like RepRap-class 3-D printers and open source microcontrollers, mass distributed manufacturing of ventilators has the potential to overcome medical supply shortages. In this study, after providing a background on ventilators, the academic literature is reviewed to find the existing and already openly-published, vetted designs for ventilators systems. These articles are analyzed to determine if the designs are open source both in spirit (license) as well as practical details (e.g. possessing accessible design source files, bill of materials, assembly instructions, wiring diagrams, firmware and software as well as operation and calibration instructions). Next, the existing Internet and gray literature are reviewed for open source ventilator projects and designs. The results of this review found that the tested and peer-reviewed systems lacked complete documentation and the open systems that were documented were either at the very early stages of design (sometimes without even a prototype) and were essentially only basically tested (if at all). With the considerably larger motivation of an ongoing pandemic, it is assumed these projects will garner greater attention and resources to make significant progress to reach a functional and easily-replicated system. There is a large amount of future work needed to move open source ventilators up to the level considered scientific-grade equipment, and even further work needed to reach medical-grade hardware. Future work is needed to achieve the potential of this approach by developing policies, updating regulations, and securing funding mechanisms for the development and testing of open source ventilators for both the current COVID19 pandemic as well as for future pandemics and for everyday use in low-resource settings.",
"keywords": [
"ventilator",
"pandemic",
"ventilation",
"influenza pandemic",
"open source",
"open hardware",
"COVID-19",
"medical hardware"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), is in part so dangerous because it threatens to overwhelm our medical infrastructure at the regional level, causing spikes in mortality rates1–4. Within the medical infrastructure, there are critical technologies that are generally available, but simply do not exist in a high enough density to handle the excessive volume of patients associated with pandemics5. Thus, people die unnecessarily throughout the world because of a combination of COVID-19 infections and the lack of access to some of these technologies6. Ventilators are an example of technologies that are currently in critical short supply7,8. Mechanical ventilators are essential for treating both influenza and COVID-19 patients in severe acute respiratory failure9,10. Past studies have shown that intensive care units (ICUs) will not have sufficient resources to treat all patients requiring ventilator support during a massive pandemic11–13, and ethically challenging triage14,15 would need to be used to decrease mortality over first-come first-served basis for ventilator allocation among patients. Some work has shown promise for using a single ventilator to support multiple patients during a disaster surge16–18. In addition, it has already been shown that 3-D printed manifolds can assist with rapidly deploying this solution and there are open source designs19. This is not necessarily straightforward20. Although some countries, like the United States, have stockpiles of ventilators21, there is consensus that there is not enough supply for serious pandemics22–25 and that rationing would be needed26. The current medical system relies exclusively on specialized, proprietary, mass-manufactured ventilators from a small selection of suppliers. This supply model clearly fails when there is a sudden surge in demand for a relatively low-volume specialty product such as ventilators in a pandemic as analyzed here. The vast majority of medical equipment is heavily patented by a few specialty medical firms that sell small volumes because during ‘normal’ times, a medium-sized hospital only needs a handful. These firms have historically aggressively protected their intellectual monopolies27–28 to the detriment of human lives (for a recent example, consider the fact that a manufacturer threatened to sue a maker for 3-D printing life-saving valves in Italy for patent infringement)29. In addition, non-practicing entities continue to attempt to actively prevent medical treatments from being deployed, even during the current COVID-19 pandemic30. Putting aside the absurdity of patenting and then obstructing others from using obvious inventions in normal times31–33, in the wake of a pandemic where millions of lives are at stake, it is intuitively obvious that this type of greed is no longer acceptable.\n\nFortunately, with the recent development and widespread deployment of open source small-scale manufacturing technologies34,35, there is now another way – mass distributed manufacturing36–39. In this new model, designs are developed and then shared with open source licenses freely on the Internet so that others can simply download and replicate the design on their own equipment, even at the household scale40. There has been tremendous and ongoing success of open source scientific hardware proliferation41–46, where lower-cost and superior-functioning custom equipment as compared to proprietary scientific tools47–50. Based on such scientific hardware results, there appears to be a significant opportunity to apply open source design principles51 and mass-scale collaborative distributed manufacturing technologies to make medical equipment52–55. In the current situation, this would at least partially overcome medical supply shortages56–61 in general, and specifically for ventilators.\n\nOf these enabling technologies, the most advanced is the fused filament fabrication (FFF)-class of desktop 3-D printers that have spawned from the self-replicating rapid prototyper (RepRap) project62–64. With the distributed manufacturing model, designs are downloaded even in remote areas and are manufactured on demand as needed65 from readily available (and possibly recycled66–78) materials. These 3-D printers are, in general, not particularly fast when making products, but with tens of thousands of 3-D printers already strategically deployed all over the world79, they have the capacity to fabricate an incredibly diverse and large range of products (growing exponentially)80, which have already been shared with open source design licenses. Here, the potential will be analyzed for hardware that can be as-much-as-possible digitally manufactured using accessible low-cost fabrication tools like RepRap-class 3-D printers and then readily constructed from widely accessible materials and simple tools (e.g. DIY hardware store sourced along with Arduino-class microcontrollers).\n\nIn this study, after providing a background on ventilators, the academic literature will be reviewed to find the existing and already openly published vetted designs for ventilator systems. These articles will be analyzed to determine if the designs are open source both in spirit (license)81 as well as required practical details, which include possessing the design source files (e.g. CAD) as well as production files (e.g. STL), PCB layouts and other electronics design files, bill of materials (BOM), list of tools required, wiring diagrams, firmware and software, as well as instructions for the assembly, calibration and operation. Next, the existing Internet and gray literature will be reviewed for open source ventilator projects and designs. Lastly, as this is a rapidly evolving area, future work will be described to enable wide-spread mass distributed manufacturing of open source ventilators to fight against the current COVID19 pandemic as well as for future pandemics and to provide the devices to low-resource regions of the world that are underserved even in normal times.\n\n\nAnalysis of literature\n\nOxygen therapy coupled with mechanical ventilation is meant to support patients so that an adequate oxygen saturation (>88%) in arterial blood is maintained82. The mechanical repository cycle has four parts: 1) inspiration, where the exhalation valve of the ventilator is closed and the ventilator uses pressured air to cause gas to flow into the lungs; 2) cycling, where changeover from inspiration to expiration occurs; 3) expiration, where the main ventilatory flow is interrupted and the exhalation valve opened to allow gas to escape from the lungs, and 4) triggering, where the changeover from expiration to inspiration occurs. According to Andreoli et al.82, mechanical ventilators are classified on what factor terminates inspiratory flow, as follows: 1) pressure-cycled ventilators terminate flow when preset pressures are reached in airways; 2) volume-cycled ventilators provide a set volume of gas to the patient over a range of pressures (but a maximum pressure is set to avoid damage to the patient’s lungs during delivery of the set tidal volume); 3) time-cycled ventilators set tidal volume by setting the inspiratory time and flow rate; and 4) flow cycled ventilators, where the inspiratory flow is terminated when the inspiratory flow rate drops below a specific level. The most common commercial modes of mechanical ventilation both provide a specified number of breaths per minute (BPM) and are 1) synchronized intermittent mandatory ventilation (SIMV) where patients can take additional breadths over the set rate and 2) assist control (AC) that uses triggering so that if the patient makes an effort to breathe, it helps them, and if not, it maintains the set rate. These modes can be used alone or in concert with 1) continuous positive airway pressure (CPAP), which uses a high-pressure reservoir and constant flow of gas that exceeds the patient’s needs; 2) positive end-expiratory pressure (PEEP), which increases the residual reserve capacity and allows for many alveoli and small airways to remain open that would otherwise close off; or 3) pressure support ventilation (PSV), which adjusts the pressure on the fly as the patient breathes to maintain a preset inspiratory pressure. For those designing open source ventilators using any of those modes and methods, there is a good base of established literature to draw upon. The classic background is available in Hess, et al.’s 1996. Essentials of mechanical ventilation,83 Tobin’s 2010 Principles and practice of mechanical ventilation84, and Owens’ 2018. The Ventilator Book85. In addition, Chapter 4 in the openly accessibly book Equipment in Anaesthesia and Critical Care: A complete guide for the FRCA, provides a good starting point to help makers understand existing designs and terminology for ventilators86. Texts area available for the use of a ventilator for the standard of care of patients with acute respiratory distress syndrome (ARDS)87, ventilator management for the NIH88, and the practical use of oxygen for patients89. A 2017 state-of-the-art review of mechanical ventilation is presented by Pham et al.90 It provides basic schematic diagrams for all of the main classes of commercialized ventilators and reviews their pros and cons.\n\nThe peer-reviewed literature itself is currently limited, but there has been some research on low-cost ventilation, even if the source is not available. First, a field portable ventilator system for domestic and military emergency medical response has been conceptually designed, but does not include enough information to construct it (e.g. the software was written in assembly language and not shared)91. This article does contain design considerations that may be useful for open source designers.\n\nA new, compact and low-cost mask respirator concept has been developed and prototyped successfully92. The blower unit was able to provide adequate ventilation to the test lungs. In addition, the integrated sensor for airway pressure was able to detect airway occlusion and leakages. It is a relatively low-power device and could be operated wirelessly with batteries. It provides a cross-sectional view of the blower unit and some details, but again, not enough to be considered full open hardware or to be easily replicated. It should be noted, however, that many of the components are within RepRap-class 3-D printing capabilities.\n\nIn addition, research has been undertaken on a pre-stage public access ventilator (PAV)93. The PAV is made up of several low cost technologies including a self-designed turbine and a range of sensors for differential pressure, flow, FiO2, FiCO2 and three-axis acceleration measurements. The PAV was tested under three conditions to show that it was adequate for an automatic emergency system: 1) pressure-controlled ventilation (PCV), 2) PCV with controlled leakage and 3) PCV with simulated airway occlusion. The PAV was tested for and showed effective ventilation for tidal volume, breathing frequency and inspiratory pressure. Similarly, there has been a proposal to replace artificial manual breathing unit (AMBU) bags with electric blowers to act as emergency ventilators94.\n\nIn contrast, another approach is to build a low-cost ventilator utilizing an AMBU bag that is not based on constant blower use95. The study by Mukaram Shahid showed the AMBU setup was able to perform all the functions of a conventional commercial ventilator for a far lower cost (<$100US excluding labor). The automated AMBU device was able to adjust the breathing rate and the volume of the air, which is comparable to older ventilators. However, it was also able to regulate the inspiration to expiration ratio and PEEP rate. Shahid’s system comes with two modes: 1) mandatory ventilation (as in older models) and 2) assisted ventilation (as with most current systems). Thus, the medical personnel can choose to use either the built-in triggering mechanism (assist boosted mode), which alters the respiration pattern once it detects a change in air pressure, or set a time interval for the respiration pattern. The article contains pictures, an electric schematic, a control loop diagram, and very basic results. Again, this can be used as starting point, but there is not enough shared to replicate in the open hardware fashion.\n\nNext, a low-cost ($420 prototype) portable mechanical ventilator was designed and prototyped that delivers breaths by compressing a conventional bag-valve mask (BVM) with a pivoting cam-actuated arm pushed by an electric motor96. This eliminates the need for a person pushing on the BVM, which is generally viewed as only a short-term solution. This system uses knobs to determine the tidal volume appropriate to the patient (usually 6–8 mL/kg of ideal body weight), adjustable BPM of 5-30, and inhalation to exhalation time ratio options of 1:2, 1:3 and 1:4 and a minimum respiratory rate97. This design is run with an open source Arduino micro-controller98 and the article provides enough details to be used as a guide for others to build a similar device, but not the full plans, code, etc. needed to qualify as an open source hardware device.\n\nThe most relevant design is a pneumatic ventilator specifically designed for pandemics, which has a low oxygen consumption99. In this study by Williams et al., they describe and test three simple, pneumatically powered, low oxygen-consumption ventilators. The three designs were tested for different lung compliances (i.e. different ventilator workloads) on the delivered FiO2 and oxygen consumption. They used a commercial mechanical test lung for these tests (Vent Aid; Michigan Instruments Inc., Grand Rapids, MI, USA). The results of this study support the potential for mass distributed production of a low-cost, gas-powered, volume-controlled ventilator with a low oxygen consumption (anywhere with oxygen at 2–4 bar). The designs could alternatively be operated on hospital compressed air. The single use, self-inflating bellows system prevents cross contamination among patients. In addition, the system possessed one-way and safety overpressure valves, which could be incorporated into other designs. The designs are in part supplied including basic principle schematics, an example BOM, but falls far short of what is expected for a complete open hardware design.\n\nThere are also completely different approaches to the design of a ventilator, such as the high-frequency oscillatory ventilator100, but only basic design schematics and preliminary testing is provided. Thus, within the peer-reviewed literature, most of the quasi-appropriate ventilator devices use a standard ventilation bag that is cyclically compressed by either an electromechanic or pneumatic setup and controlled by a microcontroller. Fortunately, the most complicated part of these designs is the controls, which is made accessible by the maturation of Arduino-based microcontrollers that can actuate and sense over a wide array of accessible and already-developed technologies (e.g. code libraries are available). It should be noted that most of the low-cost options in the literature used the bag approach, but that modern commercial ventilators are generally not manufactured with bags, bellows or pistons due to performance concerns. These concerns may be overcome by the nature of a pandemic, as well as by replacing low-cost components during failure, but this does indicate failure detection is warranted and certainly preferred in an open source ventilator design.\n\nThere are a number of proprietary commercial low-cost products like the Pumani bubbleCPAP for infants, D-box or One Breath Ventilators (not yet for sale), which could be used to relieve some of the demand for conventional ventilators. Rather than attempting to conduct a market review of such devices, however, because presumably hospitals facing a shortage of ventilators would already consider all commercially-available and regulated/approved systems, this section will investigate the growing body of knowledge to help makers develop open source ventilators as well as the preliminary designs. This section was largely supported by information gathering of the rapidly evolving open source Internet communities such as Project Open Air, which is a group of “Helpful Engineers” on the platform Just One Giant Lab. They have congregated to help in the COVID-19 pandemic by developing open source solutions and of most relevance to this study, on a project specifically on the development of open source ventilators. Their documentation and information is freely available. Although just starting, as of 17 March 2020, they have over 2,500 registered volunteers and over 9,000 on their Slack team. In addition to an offset ventilator, in their first round of project proposals, they have prioritized oxygen concentrators and personal protective equipment (PPE) as their top priority projects. In addition, their future work will focus on tube connectors and building a database for local manufacturers able to produce hardware with high score in reviews. There are other teams including those organizing around the open source wiki Appropedia for an open source ventilator. Facebook has an Open Source COVID19 Medical Supplies Group. There is a long-going Pandemic Ventilator Project that hosts their designs on Instructables. The RepRap community is starting on an open-source oxygen concentrator, which can be used alone or in tandem with an open source ventilator. Hackaday has recently called for a medical hackathon to design and deploy an open source ventilator101. Other communities are crowd-sourcing information about COVID-19 medical technologies and developing a Coronavirus Technology Handbook. Some resources for makers are appearing as basic specification provided by Botta. In addition, The Center for Safety, Simulation, and Advanced Learning Technologies (CSSALT) at the University of Florida has started an open source ventilator project based on hardware store components on the assumptions that the FDA will waive clearance for the bare-bones design if there is a massive shortage. The CSSALT system is one of the most professionally documented, with full files available for each sub-system and published engineering specifications for the ventilator that could be useful even for open hardware designers using completely different approaches. They are maintaining their documentation on GitHub. Many of the sub-modules, however, have not yet been developed, nor have a team working on them. Other projects are also using GitHub, like Jackson’s Open Respirator project, but are at the very beginning stages of development as of this writing. To assist these efforts the UK government has issued guidelines.\n\nOne approach favored by both the academic literature as well as the maker community is just to use manual ventilators – BVMs/AMBU bags. There are many commercial suppliers available and there is very preliminary documentation for open source manual ventilation for the developing world102,103. Although, in theory, purely manual ventilation could work to provide ventilation for patients over long periods, there is a real concern of both the availability of the needed man-power, as well as the continued exposure of the laborer. In addition, using a bag-valve mask may increase aerosolization of virus, and in general medical staff are not supposed to bag mask before intubation due to that risk. Many of the open source designs rely on this BVMs/AMBU bags approach where one automates the manual squeezing. It only needs an exhaust system and PEEP valve. Students at Rice University have also created an automated bag-valve mask device that fits around a normal BVM using a dual rack and pinion design with a servo motor that continuously operates (open/close) squeezing the bag a specific amount to supply air. Rice provides a full non-peer-reviewed report, that is considerably richer in details than most of the others. It offers their design strategy, a partial BOM, basic testing, the source code as well as a summary of the standards and regulations necessary to go to market. Unfortunately, in their preliminary testing, the servo motor failed after only 11 hours of service and Rice is withholding the full CAD designs and results. To overcome the limitations of both the MIT and Rice designs, a group in Ireland formed and is moving along with full open source documentation of OpenLung on GitLab. They are on their fifth iteration as of this writing based on the surrounding low-cost BVM/AMBU bag concept discussed above. Another project building off the MIT design is DIY Ventilators. Finally, the open hardware OxyGEN project is also using automated AMBU approach and although at the preliminary stages their 3-D and MATLAB design files are hosted openly on GitHub.\n\nMakers are also considering other types of non-invasive ventilators (NIV) such as those based CPAP (an alternative to PEEP), which is a form of positive airway pressure ventilator that applies mild air pressure on a continuous basis. A 3-D printed CPAP fan has been designed and tested as a blower and the design files (AutoDesk Fusion 360) and STLs are freely available. Another approach is to turn a commercial CPAP machine into a ventilator currently under development on GitHub by Lee. Lee built the system around an Arduino nano and has performed very basic tests to it that show that it provides enough pressure for a ventilator used on COVID-19 patients; however, there is not nearly enough information to recommend it for medical use. In addition, there are bi-level positive airway pressure (BiPAP) machines that are commonly used at home to treat sleep apnea and lung diseases as they decrease the effort of breathing by changing the pressure for inhalation and exhalation. Home-use BiPAPs could be used in place of hospital NIVs, but care would need to be taken because poor interfaces could generate viral aerosols104. Negative pressure ventilation (iron lung) overcomes this problem, helping lung function by pulling from the outside (there has been some development on Appropedia). It provides a full BOM, but insufficient details for replication or complete open source documentation.\n\nIn addition, several makers have developed pandemic ventilators, such as John Strupat, some time ago105, but unfortunately, in addition to the lack of testing, the source does not appear accessible. Another approach is to use a blower, as in the Pandemic Pressure Control Ventilator being developed openly on HackaDay.Io by Frank. Other open source projects are at their beginnings, like the TogRespirator project housed on GitHub developed for a Science Hackday Dublin 2020, DIY and open source respiratory and a project to build an open source ventilator on GoFundMe.\n\nIn the review of Internet-reported ventilators, it is somewhat disappointing that many of the most promising designs do not share their source code. In fact, in some cases, little more than a picture or video are available. The newer projects do tend to be following better documentation protocols. Unfortunately, despite the many promising approaches in the maker community, the one problem that the vast majority of the current partial designs have in common is that there is not nearly enough information available about their performance to recommend them for medical use.\n\nIt is clear from this review of the peer-reviewed, gray and open web literature on open source ventilators, that there is considerably more work to do. The tested and peer-reviewed systems lacked complete documentation and the open systems that were documented appropriately were either at the very early stages of design (sometimes without even a prototype) and were essentially only basically tested (and some were not tested at all). With the considerably larger motivation of an ongoing pandemic, it is assumed that these projects will garner more resources and members (as is happening with the Open Air Project) to reach a critical mass to make significant progress to reach a functional and replicable system. Although the motivation of working during a pandemic on a device that may save your life is high, the access to resources, however, is far from optimal. Already, many locations throughout the world are essentially forcing citizens to shelter-in-place, which restricts access to government and university labs, as well as to makerspaces and fab labs. In addition, some areas of the world are suffering from supply disruptions and shipping challenges. This perhaps underscores the importance of developing open source hardware for disasters before the disaster strikes. Future work is needed to develop policies and funding mechanisms for such work as it appears rational to make a small investment in developing and sharing the designs for any critical hardware.\n\nThis review article uncovered other limitations to this approach. First, due to 1) potential legal issues challenging an open source ventilator design, and 2) the general lack of useful technical information in patents (the average Instructable generally has more useful information for constructing a device than a patent despite that being a requirement of obtaining a patent), patents were not included in this review. It should be noted, however, that there are currently over 277 inactive patents in addition to those that have expired covering ventilators. Researchers can obtain this list with direct links to these patents using the Michigan Tech Free Inactive Patent Search106. There may be useful information contained in those documents that could help open source ventilator designers.\n\nAnother challenge with this approach is maintaining a proper level of sterility of devices fabricated using distributed means. Specifically, for the FFF-based 3-D printing parts, it has been reported that the prints are sterile at the time of print107. If not kept in a sterile environment, however, they could quickly become biologically contaminated. One approach to deal with this is to use washing or a chemical bath. A relatively complete analysis of the chemical compatibility of commercial 3-D printed plastics is available108. If a specific polymer is needed that cannot be 3-D printed easily, it is possible to make molds in high-temperature plastics, such as polycarbonate, and then use lower temperature plastics to make disposable single use plastic parts109. Similarly, silicone molds can be made from a 3-D printed reverse mold and used in the same way110.\n\nEven when more mature open source ventilator designs are available and can be safely manufactured by a distributed means, another area of critical future work is validation of these designs. In the medical sciences, open source devices like syringe pumps111,112 are already established113–116 and have been developed into sophisticated devices117–122. However, these devices are used in labs in general and not on people continually. For medical professionals to use an open source ventilator, they first must be convinced it will do no harm to them (or others) as well as to the patient. As COVID-19 was reported to spread via droplets, contact and natural aerosols from human-to-human, there has been a concern that high-risk aerosol-producing procedures may put medical personnel at high risk of nosocomial infections, which is a concern for some designs reviewed here123. During the airway management, enhanced droplet/airborne PPE is needed and the study by Zuo et al. provides a list of other recommendations to overcome this challenge. There have been some developments in 3-D printing some of these PPE124,125. Similarly, for designs that could aerosolize the virus, a negative pressure room would be necessary and future work is needed to design an open source approach to creating such rooms. Likewise, the greatest concern for untested open source ventilator designs is that they harm the lungs of the patients; there is significant literature in this area of ventilator-induced lung injury126–133. There are, however, solutions for preventing this, like controlling the tidal volume134,135. Thus, the designers of open source ventilators must ensure that their designs have safety features to prevent ventilator-induced lung injury, as well as having basic testing of the prototypes to ensure that the designs themselves are thoroughly vetted.\n\nWithin the open source scientific equipment community, such procedures are relatively well established and have been working reasonably well through normal peer review of hardware-based articles like those published by HardwareX, the Journal of Open Hardware, and PLOS One. For medical-equipment that could be all that stands between life and death, this vetting is even more important and open calls for papers for a Special Issue on Open-Source COVID19 Medical Hardware are attempting to address this.\n\nHowever, technical validation may not be enough. Medical hardware used on humans is also more complicated, as any studies involving humans needed to verify its functionality on people, need institutional review board approval and, if in regulated areas like the U.S., such a study would need an Investigational Device Exemption to allow for a non-FDA approved device to be used as part of a study. This is only a temporary approval and the full device would need actual FDA approval for legal deployment unless the laws are changed (or were temporarily suspended during a pandemic). These same regulatory roadblocks are in place in other nations, which has conventional ventilator manufacturers skeptical that even conventional manufacturers of other products (e.g. vacuum cleaner and automobile manufacturers are doing this now in the UK) could switch over to produce ventilators136. Clearly, this process is a problem during a pandemic. Both for the current situation and during potential future situations, there is a need to limit liability on the part of the designers, makers and users of such open source medical hardware137. Substantial future work is needed in this area. Interestingly, this gives less-developed countries with a less-formalized regulation and litigation infrastructures a distinct advantage for protecting their citizens during a pandemic by deploying open source ventilators. Although it should be pointed out that personnel and training then become the limitations to deploying mass medical efforts, even if open source ventilators are available. So, future work is needed to create training materials and translate it into the languages spoken throughout the world as well.\n\n\nConclusions\n\nThere is clear technical potential for alleviating ventilator shortages during this and future pandemics using open source ventilator designs that can be rapidly fabricated using distributed manufacturing. The results of this review, however, found that the tested and peer-reviewed ventilator systems lacked complete documentation and that the current open systems that were documented were either at the very early stages of design or had undergone only early and rudimentary testing. With the considerably larger motivation of an ongoing pandemic, it is assumed these projects will garner greater attention and resources to make significant progress to reach a functional and easily replicated open source ventilator system. There is a large amount of technical future work needed to move open source ventilators up to the level considered adequate for scientific-grade equipment and further work still to reach medical-grade hardware. Future work is needed to achieve the potential of this approach not only on the technical side, but also by developing policies, updating regulations and securing funding mechanisms for the development and testing of open source ventilators for both the current COVID19 pandemic, as well as for future pandemics and for everyday use in low-resource settings.\n\n\nData availability\n\nNo data are associated with this article.",
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Publisher Full Text\n\nZuo MZ, Huang YG, Ma WH, et al.: Expert Recommendations for Tracheal Intubation in Critically ill Patients with Noval Coronavirus Disease 2019. Chin Med Sci J. 2020; 10. PubMed Abstract | Publisher Full Text\n\nGoehrke S: 3D Printing Against Coronavirus: Who And How To Help. Fabaloo. Reference Source\n\nPrůša J: From Design to Mass 3D printing of Medical Shields in Three Days. 2020. Reference Source\n\nParker JC, Hernandez LA, Peevy KJ: Mechanisms of ventilator-induced lung injury. Crit Care Med. 1993; 21(1): 131–143. PubMed Abstract | Publisher Full Text\n\nDreyfuss D, Saumon G: Ventilator-induced lung injury: lessons from experimental studies. Am J Respir Crit Care Med. 1998; 157(1): 294–323. PubMed Abstract | Publisher Full Text\n\nSlutsky AS, Ranieri VM: Ventilator-induced lung injury. N Engl J Med. 2013; 369(22): 2126–2136. Publisher Full Text\n\nRicard JD, Dreyfuss D, Saumon G: Ventilator-induced lung injury. Eur Respir J Suppl. 2003; 42(42 suppl): 2s–9s. PubMed Abstract | Publisher Full Text\n\nDos Santos CC, Slutsky AS: Invited review: mechanisms of ventilator-induced lung injury: a perspective. J Appl Physiol (1985). 2000; 89(4): 1645–1655. PubMed Abstract | Publisher Full Text\n\nTremblay LN, Slutsky AS: Ventilator-induced lung injury: from the bench to the bedside. In: Applied Physiology in Intensive Care Medicine. Springer, Berlin, Heidelberg. 2006; 357–366. Publisher Full Text\n\nGattinoni L, Protti A, Caironi P, et al.: Ventilator-induced lung injury: the anatomical and physiological framework. Crit Care Med. 2010; 38(10Suppl): S539–S548. PubMed Abstract | Publisher Full Text\n\nCressoni M, Gotti M, Chiurazzi C, et al.: Mechanical Power and Development of Ventilator-induced Lung Injury. Anesthesiology. 2016; 124(5): 1100–1108. PubMed Abstract | Publisher Full Text\n\nBrochard L, Roudot-Thoraval F, Roupie E, et al.: Tidal volume reduction for prevention of ventilator-induced lung injury in acute respiratory distress syndrome. The Multicenter Trail Group on Tidal Volume reduction in ARDS. Am J Respir Crit Care Med. 1998; 158(6): 1831–1838. PubMed Abstract | Publisher Full Text\n\nGattinoni L, Carlesso E, Cadringher P, et al.: Physical and biological triggers of ventilator-induced lung injury and its prevention. Eur Respir J Suppl. 2003; 47(47 suppl): 15s–25s. PubMed Abstract | Publisher Full Text\n\nGompertz S, Carr E: Coronavirus: Plan to ramp up ventilator production 'unrealistic'. BBC. 2020. Reference Source\n\nKoch VG, Roxland BE: Unique proposals for limiting legal liability and encouraging adherence to ventilator allocation guidelines in an influenza pandemic. DePaul J Health Care L. 2011; 14: 467. Reference Source"
}
|
[
{
"id": "61799",
"date": "02 Apr 2020",
"name": "Richard Bowman",
"expertise": [
"Reviewer Expertise Automated microscopy and open source hardware"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a really good, thorough review of open source ventilators, including both a good technical background, a review of existing peer reviewed projects, and a summary of current efforts. I would thoroughly recommend it to anyone considering joining or using an open source ventilator project, as it's particularly good at pointing to some relevant literature that describes the requirements and principles of operation. The conclusion of the article is that we're not there yet - most of the published designs are not sufficiently complete to be easily replicated, while most of the current open projects are not rigorously tested.\n\nThe review of peer reviewed articles is interesting and does a good job of rating the different solutions in terms of openness; it is disappointing that these articles don't generally give sufficient information to reproduce the ventilator, but also unsurprising. This lends a great deal of weight to the current move towards more openness in science, where protocols, data, and schematics can be shared in data archives along with papers - but of course that's rarely done retrospectively.\n\nThe review of \"internet and gray literature\" seems objective and reasonable to me, and while such a review cannot possibly stay exhaustive given the frequency with which such projects are appearing, it does seem to cover many of the projects I've heard of. More important than an exhaustive list, however, is the discussion of the common issues to most of the DIY projects - the need for careful testing, quality control, and proper authorisation. Most discussions have focused only on technical validation - but as the author rightly points out, this is not the only way medical devices must be assessed. At least as pressing as the technical challenge is the difficulty of getting new suppliers and new devices through a quality assurance process that gives medical professionals the confidence that they can safely use said devices.\n\nOpenness is an important, and often surprisingly contentious, issue. Of the projects that are discussed, only relatively few make available complete designs for their solution. This is particularly surprising in the case of some projects from high-profile institutions that have already been widely reported in the media as \"open\" while not yet having released any designs. The commonly-accepted practice in open software is that complete designs, including source code and documentation, are made available to the public, and that a project is not considered open until this happens. Similar norms are being established for open hardware projects, supported by organisations such as OSHWA and GOSH.\n\nGiven the safety-critical nature of a ventilator, it's reasonable to be reluctant to release untested designs out of a desire to be responsible. Given the time-critical situation, sharing documentation and designs may also be considered lower priority than product development. However, the intent to share a design in the future misses the myriad benefits of open hardware - in terms of scrutiny, feedback, and improvements from the community. It also stifles the development of a community around the design, and there are many cases of promised openness never materialising. My own view is that projects ought not to claim openness until their designs are publicly available under an appropriate license, but there are definitely valid ethical and practical concerns here, and I would welcome an open debate on the best way forward.\n\nThe one statement in the article that I'm slightly troubled by is the suggestion that developing countries may be at an advantage due to their less robust regulatory systems. Firstly, while it is true that many countries in the Global South do have less formal economies, their regulations are often very tightly aligned with those in richer nations - for example, the Tanzanian medical device regulations closely mirror those used in the EU. If different standards are adhered to, it may be because the regulations are not implemented fully, rather than because the government has intentionally applied lower standards. Also, the better-resourced regulatory bodies in rich nations are more able to accelerate the process of approval if needed; it is not clear to me that a medical device would clear the bureaucratic hurdles and achieve approval any faster in a developing country, indeed the process can be much slower. It is also a very thorny ethical issue to trial medical interventions in the Global South that would not pass ethical scrutiny in richer nations, particularly as the interventions are often being proposed by people from said richer nations. I don't think the author is suggesting this, but I do feel it's a point worth highlighting. While there is often an argument made that low quality medical supplies may be better than nothing, it is also reasonable to expect that developers of technology shouldn't do anything to citizens of Low and Middle-Income Countries (LMICs) that they wouldn't do to patients in their own nation. Indeed, most ethical review panels in the UK apply exactly this criterion.\n\nThe challenge of creating a safety-critical medical device that can be produced in a distributed manner is significant, and I think the article reflects this. I could not agree more with the statement that \"technical validation may not be enough\" and would probably go further, to say that technical validation alone is not sufficient to ensure patient safety. While many open ventilator projects now exist and have gathered impressive numbers of volunteers, there remains a significant global challenge to enable such projects to be regulated appropriately, either in the current crisis or longer-term. The existing system of medical device regulation is slow, expensive, and conservative; while this conservatism has its roots in the entirely reasonable desire to prevent harm to patients, the way the system is implemented makes it extremely difficult to certify a medical device without the resources of a large company. Reform of these regulatory systems could enable a more agile approach to the design and manufacture of safety-critical components, but a satisfactory supply chain will also require significantly more quality management than is present in a typical \"maker space\" run by volunteers, hobbyists, or even experienced engineers. Questions around training and liability are also of paramount importance; while litigation against volunteers acting in good faith seems unduly harsh, there must be accountability in the supply chain of medical devices. Otherwise, we push responsibility onto the clinical staff using uncertified equipment, which adds a crippling burden to front-line staff who are already working at the limit of their capacity.\n\nOverall, I think it's right to keep an optimistic tone, while acknowledging the obvious difficulties associated with the current challenge. It's likely that, while there are many 3D printers available around the world, formal structures that do not yet exist will be needed to enable them to be fully employed to solve supply issues in this and future crises. Whether or not it is possible to make use of community designed and built ventilators in the coming months, I look forward to a world where critical supplies can be designed and produced openly for the common good. If we take the opportunity to put LMICs on a more equitable footing with respect to richer nations, the future may be more inclusive, as well as more resilient.\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": "5463",
"date": "30 Apr 2020",
"name": "Joshua M. Pearce",
"role": "Author Response",
"response": "Thank you for the thorough review and kind comments. I agree with you that the state of full open source disclosure in the general literature is disappointing and share your hope that the current move towards open science continues to accelerate. In this revised version I attempted to pull the grey literature review up to date, but as you point out that is a daily battle and I have cited the work by Read et al., trying to do that on Github. I share your surprise in the wave of what can only be described as “open washing” where high profile organizations announce their development of an open source ventilator, which upon closer inspection is not open and/or not all of the critical files have been shared. To address this, I have greatly expanded the introductory information and definition of openness as well as provided a few examples. I have also offered additional thoughts on “when to share” in the Future Work Needed Section. After considering your points and those of the other reviewers about the advantages of less developed regulatory systems I have simply removed this entirely."
}
]
},
{
"id": "61800",
"date": "21 Apr 2020",
"name": "Ramon Farre",
"expertise": [
"Reviewer Expertise Respiratory mechancs",
"Respiratory medical devices",
"Biomedical engineering."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review by Prof. Pierce is an excellent and timely piece of work. I think the author is to be commended for this panoramic perspective on the current state of the art in the open source field of mechanical ventilators for acute respiratory failure. In addition to subscribing to Prof. Bowman's extensive comments, I would particularly like to emphasize how important it is that open access contributions regarding medical devices be made in the context of multidisciplinary cooperation between professionals from different fields. In fact, unlike many other open access development areas, where the proposed devices or products can be used by a wide variety of users, medical devices focus on very specific end users: the doctors responsible for diagnosing and treating the patients. Therefore, any proposal for open access in medical devices must be not only technically sound and cost-effective, but, most importantly, safe and clinically applicable in real-life clinical routine. Consequently, as clearly stated in Professor Pierce's paper, realistically testing new developments is a critical step. Unfortunately, it is not unusual for some new open access contributions for medical applications to be the result of developments by authors drawn exclusively from the technological field, with the result that the proposed devices exhibit applicability limitations that seem obvious to clinicians who are potential users. Therefore, to advance the extension of the open access approach, it is important to make an effort to actively involve health professionals for both design and testing, trying to publish contributions in medical journals, not only in publications focused exclusively on open access. A very recent work, exactly in the field of developing open access mechanical ventilators for resource-poor areas, shows an example of a team of professionals covering the fields of biomedical engineering, respiratory physiology, physiotherapy and respiratory clinical medicine1. This multidisciplinary team has conceived, developed and tested a novel non-invasive pressure support ventilator following the conventional method of evaluating ventilators (bench tests in simulated patients and preclinical evaluation in volunteers) and has published the results in a prestigious journal of pneumology. To end my review of Prof. Pierce's excellent work, I would mention an open ethical issue about the requirements for medical devices to be used in resource-poor areas. It is important that these devices have the same high standard level as those approved for use in developed countries (e.g. CE / FDA marks). But it is also important that until the industry can provide such medical devices at affordable prices for LMICs, patients are not deprived of life-saving therapies. Finding a balance on this ethical-legal issue is difficult but fundamental.\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": "5464",
"date": "30 Apr 2020",
"name": "Joshua M. Pearce",
"role": "Author Response",
"response": "Thank you for the careful and constructive review. I have included your well-reasoned point about the need for multi-disciplinary collaboration, the need to involve medical personnel, and to aim to publish in the medical literature. In addition, I have included a deep review of Garmendia et al. design. A sincere thanks for pointing this out as well as for the work itself. Thank you for the point about ethics – to account for this I have added several points about this in the discussion."
}
]
},
{
"id": "61802",
"date": "22 Apr 2020",
"name": "Mazher Mohammed",
"expertise": [
"Reviewer Expertise Medical Devices",
"Bioengineering",
"Product Design",
"Sustainable Technologies and Materials",
"Open Source Hardware"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 The manuscript presents a review of open sources ventilators with particular emphasis towards applications in the COVID-19 pandemic during the early phases of 2020. Given the submission date of the manuscript, which has coincided with the emergence of the COVID-19 pandemic, the review is very topical, in addition to being thought provoking and insightful. COVID-19 disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), where the presenting symptoms of a patient are predominantly based upon respiratory ailments, requiring ventilation based equipment to treatment the most severe of cases. As the virus infiltrates a greater frequency of the general population, the availability of ventilation based products has rapidly become one of the primary causes for concern not only in developing, but in developed economics. Given the disruption of standard medical device supply chains during a pandemic, a viable alternative to meet the demand for ventilation equipment from healthcare providers is to turn to communities of designers, engineers, industrial specialists and knowledgeable maker enthusiasts to develop easily accessible, low-cost and open source alternatives to traditional devices. The manuscript is therefore a vital piece of documentation to assist researchers in global efforts to create ventilator alternatives. Equally, the manuscript does an exceptional job at highlighting the current state of the art in this area with balanced and considered conclusions throughout. I would therefore very much recommend this article for indexing, which will be of great interest to the scientific community and those seeking to develop their own open source solutions.\n\nRecommendations and Thoughts It is noted that the article is based on the premise of discussing ventilator technology which are deemed open source. As review manuscripts generally attract a wider target audience that scientific bodies of work, it would seem appropriate for the author to define what is meant by open source, for the benefit of unfamiliar readers. Indeed, one may see general scientific publications are open source information by virtue of the information being in the public domain. However, it is clear that the authors perception of this would require a deeper level of ‘disclosure’ of the innovations presented by various research groups to allow for ready duplication and adoption of such systems. This is starkly evident later in the manuscript during discussions of existing literature. Therefore, the distinction of what constitutes open source in this context should be explicitly defined, ideally within the introduction.\nIn light of the rapidly evolving nature of the pandemic and the volume of initiatives that are attempting to provide viable, often, open source solutions, there is likely to be some developments that have not been addressed in the review. Generally, the author has done an exemplary job of drawing the readers attention to many of the most topical and noteworthy examples. However, one would imagine as we approach the end of the pandemic period that there is likely to be a wealth of additional technologies which will have surfaced and so perhaps a follow up review may be justified, ideally inviting multiple authors involved in such projects to contribute. It is however noted that certain information within the manuscript has since evolved, specifically relating to the comment in the first paragraph of the introduction, where it is mentioned that ‘…….for a recent example, consider the fact that a manufacturer threatened to sue a maker for 3-D printing life-saving valves in Italy for patent infringement….’ Upon further development of this story, the company had made official statement to clarify that they did not attempt to sue the party which made ventilator valve parts but had primarily withheld designs based upon medical device regulation. Although withholding designs during this particular circumstance when supply chain needs could not be met resulting in potential mortality of patients, there is an argument to disclose such information despite legal implications. However, this is very much different to the notion of the company suing the Italian firm. At the time of writing the article this would not have been known to the author, but given the controversial nature of the comment, I would request the author to reword this sentence to reflect the final outcome of this case study.\nDuring the introduction when discussing the very many technologies available to the open source community, the discussion preferentially revolves around the use of rep-rap 3D printing, under the notion of digital fabrication technologies which have distributed manufacturing potential. This is a very important point to make by the author as distinctions are made as to why this approach would provide added value within the context of a pandemic. In particular, designs may be shared both at a national and international level using internet based data transfer, while leveraging manufacturing and technical capacity closer to the point of use. Such capacity has long been utilised by the open source community, providing strong resilience in instances when typical supply chains are disrupted, as would be the case during a pandemic. However, this discussion appears to be somewhat incomplete for readers who are unfamiliar with this approach of manufacturing. More specifically, it may be useful for future readers to hear some mention of other digitally driven distributed technologies, such as milling/CNC machining, laser engraving/etching and other digitally controlled tools. Indeed, several of these technologies would serve to reduce or eliminate the perceived limitations of 3D printing both in terms of manufacturable materials and speed of manufacturing. Please could the author include some additional discussion here to contextualise available options to the open source community with respect to digital and distributed manufacturing.\nThe author presents an eloquent attempt to discuss both the academic and non-academic ventilation systems, citing many interesting studies and raising most of the key facets of each respective technology. Following from the previous comments regarding the open source nature of a given study, it is highlighted that sadly many academic studies, though presenting some remarkable feats of engineering, simply do not provide sufficient information to allow for other researchers and experts to duplicate a respective ventilation system. This is indeed a shame and highlights perhaps a necessity for researchers to adopt a more open frame work of reporting academic findings and equally for reviewers to encourage and accept such ways of reporting. In light of major global health catastrophes such as pandemics, such openness within the literature may in fact prove advantageous to hasten innovations to tackle the detrimental effects on a given population.\nThe discussion on the current open source efforts appears to cover several of the major projects to the awareness of this reviewer, but more crucially provides a good cross section of important developmental aspects to inform the reader. As highlighted previously, given the rapidly evolving nature of the pandemic and the wiliness and passion of researchers and technical experts, new attempts to create ventilation products are arising on a weekly, if not daily basis from groups around the world. Therefore, in this reviewer’s opinion, to cover all such attempts would be impractical but also unnecessary given that the overlap of technical development with existing projects. As with the previous section, it would have been good to present annotated diagrams and a summary chart/table of the systems broken down into the primary attributes that fulfil the requirement for ventilation. In reading this section it was difficult to surmise how these efforts were truly moving towards a functional ventilation device. One of the more noteworthy examples from Rice University, with credible data to back the developments to date was sadly hindered by component failure after only a 11-hour evaluation period, which falls considerably short of a functional ventilator. Clearly, several if not all examples highlighted by the author raise several notes of caution toward open source design, namely the clarity and robustness of evaluation, which lacks the rigour of typical academic scrutiny in addition to the lack of standardisation of components which are suitable for purpose. Arguably, the author to varying degrees’ highlights this by stating the case studies in question are either in the very preliminary stages of investigation, show a lack of relevant performance data or a Bill of Materials (BOM) which enables scrutiny of the components employed. I believe there is a missed opportunity of discussing these elements in greater depth, which is vitally required if open source ventilators are to truly enter mainstream acceptance and use. I would very much welcome further discussion on limitations with appropriate recommendations, which both do not stifle the breadth of design ideas by the community, but also provide substantiative guidance to direct those involved to be mindful of critical milestones and ‘codes of best practise’ during the journey from inception of idea to final working and ‘usable’ ventilator. For example, looking at open source efforts for Personal Protective Equipment (PPE) we see that the 3D Printing community rapidly converged towards the preferential use of PETG polymers for manufacturing, owing to the factors of mechanical stiffness providing a semi flexible yet robust part, and importantly biocompatibility for limited human contact and being food grade to allow ease of decontamination. Equally, strict protocols were developed to minimise contamination of parts during printing, handling and shipping to both reduce the spread of the virus within the supply chain and to follow some element of best practise, similar to constraints set by medical device companies upon their manufacturing procedures. It would be very useful to the open source community if the author would share their thoughts in more explicit detail to provide a template that could be built upon for future efforts, outlining best practise from initial design ideation to working prototype. Arguably, the imperative of this is much greater than with PPE given potential intrusive nature of ventilation systems providing a clear route to internal infection through the lungs.\nOne element that appears to be missed in the present review is an overview of current commercial systems and evaluation of their performance characteristics to be both efficacious and crucially to prevent unintended harm to a patient. The author does cite an existing review of commercial ventilators by Pham et al, and so it would be unnecessary to conduct a repeat of this work. However, what would be relevant in the present manuscript is a discussion focusing on the regulatory and quality assurance aspects and how these would align and differ in the approach of open source ventilation systems. Indeed, commercial devices must adhere to the very strictest regulatory scrutiny to be classified and used as a medical device, particularly given the invasive nature of their operation to either supplement or take complete control of a patients breathing. As such there are tightly regulated frameworks of ‘fail safes’ to ensure that every component used falls within acceptable usage limits, that construction of such devices follow strict regimes for assembly and minimisation of contamination, that tests are conducted to evaluate the working performance of each device, that there is a robust training and best practise usage protocol, amongst many other safety measures and supply chain demands. Arguably, one of the biggest reservations by the commercial sector regarding open source hardware for medical device technology are the lack of process control, quality assurances and regulations regarding technological development and best practise. It would be valuable for the readers to have some insight into what safety and evaluation procedures that are conducted and how such measures would be replicated in open sources systems in general terms, highlighting key challenges which can be built upon for the future discussion.\nThe final sections of the review offer an exceptional summary of the state of art in the field of open source ventilators, highlighting several challenges and opportunities in this space. The author rightly highlights the very early stages open source ventilations systems currently are at and that we should not expect these systems to be working within a clinical setting in the immediate future. Despite this, I for one feel optimistic given the current work that is underway, our ability to access a wealth of digital knowledge, alongside the availability of hardware and manufacturing resources at our disposal. Arguably, the open source community has never been better equipment to make positive impact on the world during the COVID-19 pandemic. It is also the opinion of the reviewer that there remains many more opportunities for both growth of the open source community and to leverage the expertise synergistically with other academic groups to more rapidly advance our preparedness for emergency situations.\nReflecting upon the manuscript, I could not help but feel there are differing schools of thought that of the traditionalist and the open source innovator. The traditionalist will operate with robust scientific rigour but will provide limited information scientific manuscripts, be open to patent and potentially restrict free flow of concepts and with respect to medical technology will strictly follow medical regulatory frameworks. The open source innovator is generally driven by an overwhelming sense of openness and transparency in their work, with the belief this will help proliferate and see ideas adopted faster for anyone’s benefit, albeit on some occasions operate with a naivety towards regulatory and best practise aspects. Initial discussions by the author drew distinction between efforts of the academic and non-academic communities, which very loosely are comprised of these two types of innovators, with the exception of a few ‘maverick’ academic groups. However, there was little discussion of strategies as to how these two communities may come together in cooperation and bridge any perceived differences in thinking. Clearly the academic community prides its outcomes based on empirical evidence, the careful scrutiny of data, alongside objective design performance metrics, attributes that the authors clearly outlines as shortfalls of the general open source community. Conversely, the non-academic community provides a wealth of creativity, ingenuity, alongside technical prowess, finding often remarkable and highly efficacious solutions working with limited resources and minimal dependency of specific supply chains. I would therefore strongly welcome the author to add further discussion towards strategies on how both communities, which for the most part work independently, could align agendas to realise opportunities that transcend the sum of the two parts. Indeed, I think it more critical for the open source community, based upon the reflection of the author, to be more engaged in traditional scientific process and to incorporate this into their thinking to hasten product development for evaluation.\nOne element that gave me considerable food for thought, was the notion that developing nations have a perceived advantage during global health emergencies due to their more relaxed legal and regulatory frameworks to deploy open source ventilator systems. I am not entirely convinced that this is the case and indeed the author makes several valid arguments to the contrary, mainly that the technology is not significantly mature to function as intended and without consequence. Such suggestions can indeed have several unpleasant connotations from a legal and ethical standpoint and so I would encourage the reviewer to consider an amendment to this comment. Ultimately, I believe it was not the authors intension to imply this given previous discussions, however the context of this point should be framed better.\n\nSummary Overall, despite the sombre theme of the review, the author has done an admirable job of bringing together all the relevant themes relating to open source ventilation systems. One of biggest take home messages from the review is how much potential exists with the open source community to provide cost effective, robust and timely medical device solutions, which may be far less susceptible to supply chain disruption and leverage a greater capacity for localised fabrication using the distributed manufacturing model. This capacity can only be realised by continued development of existing open source projects, increased dialogue with academic groups to work collaboratively to validate and iteratively improve ventilation system concepts for maximum efficacy. Equally, there is a clear need for regulatory reform which appreciates the evolving circumstances during a global health crisis and could provide an alternative framework to leverage capacity outside of typical medical supply chains to supplement efforts on the ground, as and when appropriate. What this framework should look like is another debate entirely, but this article makes an elegant argument for the debate to be had.\nDespite the infancy of open source ventilation systems, much potential exists and it is an exciting time for developers to continue their efforts towards working solution. It is exciting to see what may be a paradigm shift in how we perceive and operate globally in the medical device sector, particularly in light of the recent issues during the COVID-19 pandemic, which have decimated supply chains, while the shear volume of cases has put a drain on medical resources. The pandemic has already seen the use of open source designs, manufactured in a distributed manner, make impact to supplement shortfalls in PPE equipment. Could the same in time be true for ventilator technologies based on the balance of growing demand and available resources? Only time will tell. I reiterate the relevancy of the article by the author and the manuscript has been a very thought provoking document to read and digest. I would whole heartedly recommend this article for publication and encourage researcher and technologist in the field to draw inspiration from the insightful and thought provoking arguments outlined. I do welcome a time when the lessons we are learning during the pandemic lead to a more caring and equitable world for us all, and it feels from a technological standpoint, that open source innovation will be part of that story.\n\nAdditional minor points for consideration\nThe hyperlink for Ref 82 needs revising as it links to an error page.\n\nOne the first paragraph of page 4 ‘breadth’ has mistakenly been used in place of ‘breath’. Additionally there are some minor grammatical errors toward the end of this same paragraph that need amending, specifically the sentence starting with ‘texts area available for the ……..’\n\nGenerally speaking, it would have been very useful to contextualise much of the discussion with annotated diagrams of several key open source innovations, to give the reader a real feel for the types of devices in development. Indeed, this is typical of many academic based reviews of the scientific and gray literature. Unfortunately, this has not been the case in the present manuscript and I would invite the author to consider such an amendment.\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": "5465",
"date": "30 Apr 2020",
"name": "Joshua M. Pearce",
"role": "Author Response",
"response": "Thank you for the rigorous and exceptionally detailed review. Following your recommendation, I have added a much more explicit section detailing the meaning of open source as well as provide examples of it being misused. I have removed the now incorrect example of the manufacturer suing over reverse engineered valves. Although it may be interesting to note I contacted the individual who reversed engineered them before the initial article was published and I have yet to receive the STL files. I share your frustration with the current lack of appropriate sharing. I have included a more detailed review of the existing designs – but as you point out it is impractical to do everything in such a rapidly changing field where websites are being updated hourly or faster. I have declined to develop diagrams - even if I could find appropriately licensed images as a summary because of this rapidly changing nature. Particularly in mechanical designs because those appear to be the most often altered. I have expanded considerably what the files are for in open hardware to help better introduce those outside of the open hardware community to the concept as well as make it clearer why they need to be included. I have brought in an example from the PPE community. In addition, following your recommendation I have attempted to provide some best practices in terms of ‘when to share’ in the discussion. This is meant to bridge the gap between the traditionalist and maker philosophies you discuss. After considering your points and those of the other reviewers about the advantages of less developed regulatory systems I have simply removed this entirely. I have corrected the minor mistakes you pointed out – thank you for finding them. Lastly, I don’t think that a complete review of all the legal hurdles and regulatory framework needed in this technical space can be done here - it needs a completely separate review. We have a major problem here because even some of the standards are not available. When an artificial lung company contacted their customers on my behalf to get a testing protocol they referred me to a long list of ASTM standards which I was not able to acquire through my relatively-well-resourced University library. I purchased the first one and was disappointed to find that it mostly contained references to other standards and a shocking dearth of useful technical information. As there is a conflict of interest between the publishers of such standards and providing all the information in a single location that is freely available for designers to help in a pandemic, this represents one of the largest hurdles. Some good-willed publishers have provided open access to some of their documents during the pandemic. This is a nice start but a better approach is to use public funding to provide open access documentation as the cost and opacity of these standards provide a barrier to open source ventilator development and as I pointed out an unacceptable state during an emergency."
}
]
}
] | 1
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https://f1000research.com/articles/9-218
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https://f1000research.com/articles/8-56/v1
|
14 Jan 19
|
{
"type": "Research Article",
"title": "Metallothionein expression on oysters (Crassostrea cuculata and Crassostrea glomerata) from the southern coastal region of East Java",
"authors": [
"Asus Maizar Suryanto Hertika",
"Kusriani Kusriani",
"Erlinda Indrayani",
"Defri Yona",
"Renanda Baghaz Dzulhamdhani Surya Putra",
"Kusriani Kusriani",
"Erlinda Indrayani",
"Defri Yona",
"Renanda Baghaz Dzulhamdhani Surya Putra"
],
"abstract": "Background: This study aimed to analyse levels of heavy metals (Pb, Hg and Cd) in the aquatic body, gills and stomach of the oysters Crassostrea cuculata and Crassostrea glomerata, the metallothionein (MT) level in the gills and stomach of both oysters, and relationships between heavy metals level (Pb, Hg and Cd) in the gills and stomach to MT level in both types of oysters. Methods: The research method utilized was a descriptive method. The oyster samples were taken from three stations: Sendang Biru, Popoh and Prigi beaches. MT values were assessed using confocal laser scanning microscopy. The heavy metal levels were assessed using atomic absorption spectrophotometry method. Results: Both oyster heavy metal content obtained in the southern coastal waters exceeded the safe limit set by the State Minister of Environment No. 51 of 2004. In general, the expression of MT was found to be higher in gastric tissue compared to gill tissue. Conclusions: The relationship between levels of the heavy metals Pb, Hg, and Cd in oyster gills and stomach has a strong relationship with MT levels in the gills and stomach in both types of oysters.",
"keywords": [
"Heavy Metal",
"Biomarkers",
"Metallothionein",
"CLSM"
],
"content": "Introduction\n\nCoastal areas are often under the pressure of ecological pollution originating from human activities. One kind of pollutant is heavy metals, such as cadmium (Cd), Mercury (Hg), and lead (Pb), which originate from household and industrial waste effluent1,2. Heavy metals settle on the bottom of the seabed by sedimentation. This can contaminate marine biota with heavy metals and threaten human health as consumers3–7.\n\nMetallothionein (MT) is a non-enzymatic protein in a low molecular weight which has a high cysteine content, does not have aromatic amino acids and is not heat-stable. The multiple thiol groups (-SH), formed by cysteine residues, allow MT to bind heavy metals8–10. MT has a specific metal binding ability. Each MT only binds one type of metal, with Cd M, Hg and Pb each binding a different MT11,12. MT has been widely used as a specific biomarker because the expression of MT reflects the presence of heavy metals13–15. Previous research has revealed that the induction of MT expression increases after the organism is exposed to heavy metals16. Hertika et al.17 found the existence of positive relationships between heavy metals and MT expression in North East coast oysters.\n\nMT possess the ability to bind a certain amount metal in a cell and restore the ability to function of inactive proteins due to metal cadmium18. According to Prabowo19, heavy metals contained in waters can enter the body of aquatic biota. Heavy metals pass through the mouth and digestive organs, such as the surface of the gills. Therefore, organisms that live in waters with higher levels of heavy metal contamination will have higher heavy metal level.\n\nOysters, including benthic macrofauna species, are one of the best bioindicators of heavy metal contamination in an area20. Oysters are potential biota contaminated by heavy metals, as these are filter feeders, and express MT, which is able to bind heavy metals Therefore, oysters can be used as test animals in monitoring the accumulation of heavy metal levels in polluted waters21. This study aims to analyze heavy metal level (Pb, Hg, and Cd) in the tissues (gills and stomach) of oysters (Crassostrea cuculata and Crassostrea glomerata) and the coastal waters of the South coast of East Java to determine their relationship to MT (MT) expression.\n\n\nMethods\n\nIn total, three samples of oyster (Crassostrea cuculata and Crassostrea glomerata) were collected in three of each of the three sub-stations on the Sendang Biru (Malang) coast, Prigi beach (Trenggalek) and Popoh beach (Tulungagung). Sub-station 1, 2 and 3 on the Sendang Biru beach are geographically located at 8°26’01.3”S 112°41’01.8”E, 8°26’04.7”S 112°40’55.3”E and 8°25’48.2”S 112°41’17.0”E, respectively. Sub-stations 1, 2 and 3 of the Prigi coast are geographically located at 8°25’48.2”S 112°41’17.0”E, 8°15’47.9”S 111°48’11.6”E and 8°15’44.4”S 111°48’13.0”E, respectively. Sub-stations 1, 2 and 3 from Popoh beach are geographically located at 8°17’11.9”S 111°43’41.9”E, 8°17’13.2”S 111°43’47.2”E and 8°17’11.8”S 111°43’33.1”E, respectively. Oyster samples were taken three times for gills and gastric tissue taken in each sub-station and each was analysed separately.\n\nHeavy metals (Pb, Cd, and Hg) in oysters (gills and gastric tissue) and the seawater at each sub-station were measured by atomic absorption spectrophotometry (AAS) following the measurement procedures in previous studies carried out by Hertika et al., 201817 A total of 50 ml seawater samples obtained from each substation were filtered with a 0.45-mm polycarbonate membrane to separate particles which caused contamination in heavy metal measurements. Next, 1 M nitric acid was added to the water sample to obtain a pH value below 2.\n\nThe gill tissue and stomach taken from the oyster samples in each substation were prepared according to the method of Trinchella et al.22. In order to obtain a complete oxidation process in the decomposition of organic substances, to each sample of gill and gastric tissue (0.2 grams), 2 ml of HNO3 were added. The samples were incubated for 30 minutes at low temperatures (5-8°C) to avoid minerals lost during the evaporation process. The sample is centrifuged for 15 minutes at 12,000g. The supernatant produced from the centrifugation process was taken to measure the heavy metal content. Measurement of heavy metals (Pb, Cd, and Hg) was carried out using the A220 Atomic Absorption Spectrophotometer Variant (Variant, Inc.).\n\nTo analyse MT expression in this study, confocal laser scanning microscopy (CLSM) was performed (Confocal Olympus FluoViewTM FV1000) based on previous research by Ockleford23 and Mongan et al.24. This observation system utilized a reverse-light-path fiber-optic signal that transmits Nomarski DIC signals to a second detector to visualize immunofluorescent and refractive index (RI) images. Images were observed using an Olympus U-TBI90 Microscope (Olympus, Japan) and inputted to Olympus Fluoview v4.2a, Japan for calculating MT expression quantities. Briefly, the gills and stomach in oyster samples (Crassostrea cuculata and Crassostrea glomerata) were preserved into 10% formaldehyde. The sample was cut into 2-3 mm sections using a microtome and dehydrated using the Tissue Tex Processor. The samples were twice soaked with xylol (#CAT 1086612511, Merck, Japan) for 10 minutes each. Then the sample was fixed with absolute ethanol 90%, for 5 minutes. Immediately, the sample was soaked with 10 mM pH 6 buffer citrate for 15 minutes. Samples were blocked with PBST containing BSA 2% (CAT# 15561020, Thermo Fisher, USA) for 1 hour in room temperature. Furthermore, the sample was labelled with the Anti-MT Primary monoclonal mouse Antibody (1:1000, CAT# UC1MT, Gene Tex, USA) which contained 2% BSA for 1 hour at room temperature. Samples were rinsed with PBST for 8 minutes. Furthermore, the sample is labeled with rhodamine-conjugated Mouse IgG Antibody (CAT#610-1002, ROCKLAND Immunochemical Inc,USA) containing 2% BSA for 1 hour at room temperature. The sample was rinsed using PBST and dried. Lastly, glycerol was added into the sample and observed using CLSM.\n\nWater quality in this study was measured based on standard methods25. Dissolved oxygen concentration was measured in situ using an oximeter (YSI PRO 20). The pH was also measured in situ using a pH pen (PH 2011 ATC) at each sub-station. Temperature and salinity were measured using a mercury thermometer and refractometer (RHS-10ATC, SINOTECH), respectively.\n\nThis study used regression correlation analysis with a simple linear regression model in SPSS version 16.0 software. Professional charts are created using the GraphPad Prism 7.00 application. Using the method outlined bn Hertika et al.17, the relationship between heavy metal levels with MT (MT) expression was obtained from multiple regression results with variable Y exhibiting heavy metals in oyster gills or gastric tissue. Variable X exhibited levels of Pb, Cd, and Hg.\n\n\nResults and discussion\n\nAnalysis of the Pb, Cd and Hg content at the three research stations (Sendang Biru, Popoh, Prigi) is shown in Figure 1. The range of highest Pb concentrations in each substation was 0.03–0.054 mg l-1; the highest Hg concentration was 0.01–0.026 mg l-1 and that of Cd was 0.009–0.018 mg l-1. The highest concentration of Pb, Cd and Hg heavy metals were found at sub-station 2 in Sendang Biru for 0.054, 0.018, 0.026 mg l-1. In general, some measured heavy metal content has passed the specified quality standard. Based on the Decree of the State Minister of Environment No. 51 of 2004 concerning seawater quality standards for heavy metal content26, the Hg content appropriates the aquatic environment must not exceed 0.003 mg l-1, Pb 0.05 mg l-1 and Cd 0.01 mg l-1.\n\nHeavy metals (Pb, Cd, and Hg) in Crassostrea cuculata and Crassostrea glomerata gills and gastric tissue is exhibited in Figure 2. The highest Pb content in the gills of Crassostrea cuculata was obtained at the Prigi station, at sub-station 1 at 0.13 mg l-1, the highest Cd and Hg concentrations were obtained from Sendang Biru station in sub-station 2 at 0.08 mg l-1 and 0.09 mg l-1, respectively (Figure 2A). Whereas the highest heavy metal level in Crassostrea cuculata gastric tissue Pb, Hg and Cd levels were observed at the Sendang Biru station in sub-station 1, at 0.067, 0.036 and 0.077 mg l-1 respectively (Figure 2B).\n\nPb, Cd, and Hg levels in the gills and stomach of (A, B) Crassostrea cuculata and (C, D) Crassostrea glomerata at Sendang Biru, Popoh and Prigi stations.\n\nFurthermore, the heavy metal content in the gills of Crassostrea glomerata is exhibited in Figure 2C. The highest Pb, Hg, and Cd values were obtained at Sendang Biru station at sub-station 1, at 0.142, 0.071 and 0.11 mg l-1, respectively. The highest value of gastric Pb and Cd content was observed in Prigi stations in substations 1 and 3: 0.145 and 0.047 mg l-1, respectively (Figure 2D). The highest value of Hg was obtained at Sendang Biru station at sub-station 1, which was 0.078 mg l-1.\n\nThe accumulation of heavy metals in this study exhibit the same pattern in the study conducted by Bilgin et al.27, which found that accumulation of heavy metals in the soft tissue of mollusks, from highest to lowest, is Pb> Cd> Hg. Aquatic organisms are capable of absorbing and accumulating heavy metals in several ways: through the respiratory tract (gills), digestive tract, and skin surface diffusion28. In this study, the highest accumulation of heavy metals was found in gill tissue. According to Hutagulung29, the high accumulation of heavy metals in gills is closely related to the nature of biota. The oyster’s food intake is conducted through filtering water (filter feeders). Furthermore, Soto et al.30 revealed that gills are the main target tissue for absorption contamination of dissolved heavy metal ions in aquatic bodies.\n\nResearch result exhibited that Crassostrea cuculata and Crassostrea glomerata have different values of heavy metals as each organism has a different ability to accumulate heavy metals. Based on the results of the study by Fattorini et al.31 Mytilus galloprovincialis is able to accumulate Pb, Cd and Hg heavy metals at 0.29–2.95 mg l-1, 0.41–1.60 mg l-1, and 0.02–0.19 mg l-1, respectively. However, Kucuksezgin et al.32 achieved different results; Thylacodes decussatus was observed to absorb Pb at levels ranging from 0.38–1.2 mg l-1, Cd at 0.03–0.24 mg l-1, and Hg at 0.04–0.13 mg l-1. This may be related to the tendency of specific bioaccumulation of bivalves, based on different habitats, lifestyles, and abundance of food. Some studies emphasize that metal accumulation has presented different species-specific capacities for bivalves33,34. It is claimed that this difference is related to the metabolic rate of bivalve species35. The bioaccumulation pattern of metals can generally be attributed to the presence of anthropogenic inputs or lithogenic sources affecting the area. Seasonal variations in bivalves metal concentrations result from many factors, such as large differences in water temperature, particulate metal runoff to coastal waters, food availability. It is caused by transferring metals from water to feeding-filtering organisms, body weight changes during gonadal development, and biomass release associated with sexual reproduction36–38. For bivalves, the accumulated changes depend on the metal and the ability of different species or genera to store or/and remove metals from the tissue. In general, metal concentrations in bivalves increase with increasing shell size; however, in some cases, metal concentrations may decrease due to the detoxification process in these organisms39,40. Raw data are available on OSF41.\n\nMT content analysis is exhibited in Figure 3. Research results exhibited that in MT gastric tissue overall expression was higher than gill tissue (Figure 3A). The highest metallothionine expression in gill tissue was obtained at Prigi station at 810.876–1387.61 arbitrary units. The lowest expression on the gastric tissue was found at Sendang Biru station ranging from 453.246–511.098 arbitrary units. Highest MT expression in gill tissue was also found at Prigi station ranging from 325.976–622.534 arbitrary units. On the other hand, the lowest value was obtained at the Sendang Biru station at 276.254–498.512 arbitrary units. MT expression in the stomach tissue is higher than that in the gill tissue. This is supported by assessment of the morphology of MT expression using rhodamine-labelled MT in the gill and stomach tissue. In Figure 3B, Rhodamine-MT as metallothionine marker is expressed brighter in gastric tissue compared to gill tissue. Figure 3C shows rhodamine-MT absorption, as an MT marker, is recorded to have a higher intensity in gastric tissues compared to gill tissues.\n\nMetallothionein (MT) expression in Crassostrea cuculata gills and stomach. (A) The quantity of MT expression at each station. (B) Morphology of MT expression in gills and stomach, (C) Absorbance gating of MT expression from a representative experiment.\n\nHeavy metal content was inversely proportional to MT expression in the gill and gastric tissues. The high content of heavy metals in the gills stimulates high MT expression to bind and detoxify heavy metals quickly. Therefore, MT quantity detected on the tissue decreases. Conversely, heavy metal in gastric tissue accumulates less compared to gill tissue. Hence, the detoxification process is slower. It indicates that MT quantity detected in this study is higher. According to Ringwood et al.42, there is a positive relationship between MT and heavy metal pollutants. Heavy metal contaminants can cause systemic damage to an organism and result in increased MT production. Previous research has revealed that MT has a crucial role in various processes of biological activity; it binds heavy metals and conduct recovery process from systemic damage caused by heavy metals through homeostasis process (dynamic balancing of the body's biological processes) to heavy metals43,44, and heavy metal detoxification45,46. The function of MT in heavy metal detoxification mainly depends on the high-affinity bond between heavy metals and MTs, which causes heavy metal absorption to be higher than that of important macromolecules45,47. It indicates that MT plays an important role in protecting cells from heavy metal poisoning48–51. It is proven that MT could be a biomarker useful for predicting heavy metal toxicity and heavy metal detoxification toxic to organisms52,53. Raw data are available on OSF41.\n\nMT content analysis is exhibited in Figure 4. Similar results were obtained in the analysis of Crassostrea glomerata gills and stomach. MT expression in the gastric tissue of Crassostrea glomerata is expressed higher than gill tissue (Figure 4A). The highest MT expression in gastric tissue was obtained at Prigi Station sub-station 1 with a value of 1412,112 arbitrary units. The lowest MT expression in gastric tissue was obtained from Sendang Biru sub-station 3 with a value of 576,243 arbitrary units. Furthermore, the highest MT expression in gill tissue was obtained from Prigi substation 1 with a value of 756,381 arbitrary units. The lowest MT tissue gill expression was obtained at Sendang Biru substation 3, with a value of 366,125 arbitrary units. Higher MT expression was observed in gastric tissue compared to gill tissue morphologically (Figure 4B). The morphological results exhibited that MT labeled Rhodamine-B in gastric tissue appears brighter than gill tissue. Rhodamine-MT is a MT marker used in this study. Figure 4C exhibited that the Rhodamine-MT absorption as an MT marker possesses a higher intensity in gill tissue compared to gastric tissue.\n\nMetallothionein (MT) expression in gills and stomach of Crassostrea cuculata. (A) The quantity of MT expression at each station. (B) Morphology of MT expression in gills and stomach. (C) Absorbance gating of MT expression from a representative experiment.\n\nRumahlatu, et al.18 stated that MT protein which acts as a metal binding protein can be used as an indicator of pollution, as the presence of MT in oysters serves as a binder of heavy metals that accumulate in the body. Based on the research result, MT expression in Crassostrea glomerata and Crassostrea cuculata has different results. According to Tapiero and Tew54, MT expression levels vary between species, these levels are determined by the identity of metal atoms bound to proteins, and the difference in metal distribution between MT isoforms. This may affect MT expression levels, therefore indicating that MT is involved in cellular homeostatic control and element regulation. MT expression in Crassostrea cuculata highest value was found in gastric tissue and the lowest value in gill tissue. This is inversely proportional to the heavy metal content, which wsa highest in the gills. In this case, the high content of heavy metals in the gills causes high MT production, which in turn is rapidly used for homeostasis and detoxification from damage caused by these heavy metal toxins. Therefore, MT expression in the gill tissue was detected as lower than that in gastric tissue. In previous studies, MT participated in metal ion homeostasis and detoxification, and anti-oxidative damage55–57. Furthermore, MT expression is governed by the rate of accumulation of heavy metals, and MT plays an important role in metal detoxification and homeostasis58,59. Some species develop physiological adaptations to tolerate metal pollutants60 which use two major detoxification mechanisms. The oyster uses metal binding compounds in the cytosol, such as MT (or similar proteins), or mineralization of minerals61. The relativity of these two detoxification mechanisms varies greatly depending on the species and habitat. According to Amiard et al.61, a decrease in MT concentration in organisms accumulated by heavy metals is influenced by cytotoxic effects in the detoxification process. Should an organism accumulate high heavy metals, a significant reduction in MT is caused as it is used in the process of suppressing the reactive production of ROS species oxygen responsible for oxygen metabolism62.\n\nThe relationship between heavy metal level Pb, Hg, and Cd with MT levels in Crassostrea cuculata gills exhibited a very strong value with the coefficient of determination (R2) of 0.908. Based on the results of multiple linear regression equations of heavy metal level in the aquatic body against MT levels in Crassostrea cuculata gill tissue, the following formula was used Y = 242.337 + 2,128.234 X1 + 88.354 X2 + 2,182.218 X3. These results indicate that a 1 ppm Pb (X1) increase will increase MT expression 2,128,234 arbitrary units. Should Cd increased by 1 ppm (X2), it will increase MT expression by 88,354 arbitrary units. On the other hand, a 1 ppm Hg (X3) increase would increase the MT expression of 2,182,218 arbitrary units.\n\nFurthermore, a similar result was found in the relationship between the heavy metal level of Pb, Hg, and Cd with the gastric tissue MT expression. It indicates a strong relationship with the value of the coefficient of determination (R2) of 0.92. Multiple linear regression equations of heavy metal level in the aquatic body against MT levels Crassostrea cuculata gastric assessment obtained the equation Y = 494.528 + 4,075.811 X1 + 2,852.821 X2+ 5,990.359 X3. The equation exhibited that Pb (X1) 1 ppm increase would, in turn, increase MT expression 4,075,811 at arbitrary units. Cd (X2) 1 ppm increase would increase MT expression 2,852,821 at arbitrary units. Hg (X3) when rising 1 ppm increase will increase MT expression of 5,990,359 arbitrary units.\n\nHasan et al.63 stated that when the accumulation of heavy metals in the body of shellfish increases the synthesis of MT will probably reach the maximum level. The research conducted by Li et al.64, exhibited a positive correlation between Cd heavy metal and MT levels in the gills and mantle of the bivalve group, which means that MT can be used as a biomarker for Cd heavy metal pollution. Furthermore, Sakulsak et al.65 stated that the occurrence of exposure to heavy metals and the accumulation of heavy metals in cells can increase MT levels in tissues. Hence, MT can be used as a biomarker in environmental toxicology.\n\nA very strong relationship was obtained in Pb, Hg, Cd heavy metal level and gill MT expression of Crassostrea glomerata with a coefficient of determination (R2) of 0.943. Multiple linear regression equations of heavy metal level in aquatic body against MT expression of Crassostrea glomerata gills is Y = 320.254 + 2,311.778 X1 + 910.719 X2 + 2,173.765 X3. This equation exhibited that a 1 ppm increase in Pb (X1) will increase MT expression 2,311,778 arbitrary units. Furthermore, a 1 ppm increase in Cd (X2) will increase MT expression 910,719 arbitrary units. A 1 ppm increase in Hg (X3) will cause an increase in MT expression at 2,173,765 arbitrary units.\n\nThe relationship between the heavy metal level of Pb, Hg, Cd in the aquatic body and MT expression Crassostrea glomerata gastric tissue exhibited a strong relationship with the value of the coefficient of determination (R2) of 0.918. Multiple linear regression equations of heavy metal level in the aquatic body against MT expression in Crassostrea glomerata gastric tissue was found to be similar. Y = 570.492 + 4,603.743 X1 + 3,455.676 X2+ 4,333.870 X3. The equation exhibited Pb (X1) up 1 ppm increase will cause an increase in MT expression of 4,603,743 arbitrary units. Furthermore, a 1 ppm increase in Cd (X2) will cause an increase in MT expression of 3,455,676 arbitrary units. A 1 ppm increase in Hg (X3) will result in an increase in MT expression of 4,333,870 arbitrary units.\n\nAccording to Rumahlatu, et al.,18, MT acts as a metal-binding protein. It can be used as an indicator of pollution, as the presence of MT in oysters serves as a binder of heavy metals that accumulate in the body. Although many species can produce MT, oysters have exhibited a higher accumulation rate for metals compared to other species because they are filter feeders and tend to settle in one place66. MT can bind metals very strongly, but exchanging bonds with other proteins may take place easily. MT bonds to metals possess high thermodynamic stability but low kinetic stability67.\n\nWe observed physical and chemical water quality parameters that support the life of Crassostrea cuculata and Crassostrea glomerata, namely temperature, acidity (pH), dissolved oxygen (DO) and salinity (Figure 5).\n\nWater quality monitoring exhibited that there is no significant difference between each station and exhibited that the water quality is good for the oyster ecology. According to KEPMENLH. 51 of 200426, it indicates the temperature suitable for oysters growth is 25-34°C. Furthermore, the pH level suitable for oysters’ ecosystem ranging from 6.8 to 8.8. DO levels of more than 5 mg l-1 are required to support aquatic organisms’ survival26.\n\n\nConclusion\n\nBased on the results of the study it can be concluded that the heavy metal levels in the three locations assessed (Sendang Biru, Popoh, and Prigi) have exceeded the specified quality threshold. Furthermore, the relationship between Pb, Hg, and Cd heavy metal level in the aquatic body has a strong relationship with the expression of MT in oysters’ stomach and gills (Crassostrea cuculata and Crassostrea glomerata).\n\n\nData availability\n\nRaw data from the present study, including heavy metal levels in all oyster samples and all raw immunofluorescent images, are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/37BVQ41.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nFunding for this study was provided by the General Directorate of Research and Development, Ministry of Research and Technology and Higher Education, Research Contract, Number: 063/SP2H/LT/DRPM/IV/2017.\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 hereby wish to acknowledge the Central Laboratory of Life Science (LSIH), University of Brawijaya for granting us permission to carry out this work using their CLSM analysis instrument.\n\n\nReferences\n\nEmersida I, Sukendi S, Amin B: Kandungan Logam Berat Pada Air Dan Tiram (Crassostrea cucullata born) di Muara Sungai Loskala Kota Lhokseumawe Provinsi Aceh. Berkala Perikanan Terubuk. 2014; 42(1): 69–79. Reference Source\n\nVélez D, Montoro R: Arsenic speciation in manufactured seafood products. J Food Prot. 1998; 61(9): 1240–1245. 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PubMed Abstract | Publisher Full Text\n\nHertika AMS, Kusriani K, Indrayani E, et al.: Relationship between levels of the heavy metals lead, cadmium and mercury, and metallothionein in the gills and stomach of Crassostrea iredalei and Crassostrea glomerata [version 1; referees: 2 approved]. F1000Res. 2018; 7: 1239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRumahlatu D, Corebima AD, Amin M, et al.: Kadmium dan Efeknya terhadap Ekspresi Protein Metallothionein pada Deadema setosum (Echinoidea; Echinodermata). Jurnal Penelitian Perikanan. 2012; 1(1): 26–35. Reference Source\n\nPrabowo R: Akumulasi Kadmium Pada Daging Ikan Bandeng Accumulation Cadmium Of Bandeng Fish Flesh. Mediagro. 2005; 1(2): 58–74. Reference Source\n\nIrnidayanti Y, Septiana S, Layli FA: Metallothionein gene expression in green mussels (Perna viridis) as a bioindicator for heavy metal on the North Coast of Java and its impact on the development of reproductive organs. In World Congress of Reproductive Biology. BioScientifica. 2014; 1: 312. Publisher Full Text\n\nWulandari E, Herawati EY, Arfiati D: Kandungan logam berat Pb pada air laut dan tiram Saccostrea glomerata sebagai bioindikator kualitas perairan Prigi, Trenggalek, Jawa Timur. Jurnal Penelitian Perikanan. 2012; 1(1): 10–14. Reference Source\n\nTrinchella F, Esposito MG, Simoniello P, et al.: Cadmium, lead and metallothionein contents in cultivated mussels (Mytilus galloprovincialis) from the Gulf of Naples (Southern Italy). Aquaculture Res. 2013; 44(7): 1076–1084. Publisher Full Text\n\nOckleford C, Editorial.: The confocal laser scanning microscope (CLSM). J Pathol. 1995; 176(1): 1–2. PubMed Abstract | Publisher Full Text\n\nMongan LC, Gormally J, Hubbard ARD, et al.: Confocal microscopy: theory and applications. In: Lambert, editor. Calcium signalling protocols. Totowa, NJ: Humana Press. 1998; 1–39.\n\nAPHA: Standard methods for the examination of water and wastewater. 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PubMed Abstract | Publisher Full Text\n\nFattorini D, Notti A, Di Mento R, et al.: Seasonal, spatial and inter-annual variations of trace metals in mussels from the Adriatic sea: a regional gradient for arsenic and implications for monitoring the impact of off-shore activities. Chemosphere. 2008; 72(10): 1524–1533. PubMed Abstract | Publisher Full Text\n\nKucuksezgin F, Kacar A, Kucuksezgin G, et al.: Monitoring metal contamination levels and fecal pollution in clam (Tapes decussatus) collected from Izmir Bay (Turkey). Environ Monit Asses. 2010; 162(1–4): 407–415. PubMed Abstract | Publisher Full Text\n\nUsero J, Gonzalez-Regalado E, Gracia I: Trace metals in the bivalve molluscs Ruditapes decussatus and Ruditapes philippinarum from the Atlantic coast of southern Spain. Environ Int. 1997; 23(3): 291–298. Publisher Full Text\n\nIrato P, Santovito G, Cassini A, et al.: Metal accumulation and binding protein induction in Mytilus galloprovincialis, Scapharca inaequivalvis, and Tapes philippinarum from the Lagoon of Venice. Arch Environ Contam Toxicol. 2003; 44(4): 476–484. PubMed Abstract | Publisher Full Text\n\nNajdek M, Sapunar J: Total and methyl-mercury content in bivalves, Mytilus galloprovincialis Lamarck and Ostrea edulis Linnaeus: relationship of biochemical composition and body size. Bull Environ Contam Toxicol. 1987; 39(1): 56–62. PubMed Abstract | Publisher Full Text\n\nSzefer P, Kim BS, Kim CK, et al.: Distribution and coassociations of trace elements in soft tissue and byssus of Mytilus galloprovincialis relative to the surrounding seawater and suspended matter of the southern part of the Korean Peninsula. Environ Pollut. 2004; 129(2): 209–28. 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PubMed Abstract | Publisher Full Text\n\nHertika AMS: Metallothionein Expression on Oysters (Crassostrea cuculata and Crassostrea glomerata) from the southern coastal of East Java. 2018. http://www.doi.org/10.17605/OSF.IO/37BVQ\n\nRingwood AH, Hoguet J, Keppler C, et al.: Linkages between cellular biomarker responses and reproductive success in oysters--Crassostrea virginica. Mar Environ Res. 2004; 58(2–5): 151–155. PubMed Abstract | Publisher Full Text\n\nDe Lisle RC, Sarras MP Jr, Hidalgo J, et al.: Metallothionein is a component of exocrine pancreas secretion: implications for zinc homeostasis. Am J Physiol. 1996; 271(4 Pt 1): C1103–C1110. PubMed Abstract | Publisher Full Text\n\nPearce LL, Wasserloos K, St Croix CM, et al.: Metallothionein, nitric oxide and zinc homeostasis in vascular endothelial cells. J Nutr. 2000; 130(5S Suppl): 1467S–1470S. PubMed Abstract | Publisher Full Text\n\nHuang PC, Morris S, Dinman J, et al.: Role of metallothionein in detoxification and tolerance to transition metals. Experientia Suppl. 1987; 52: 439–446. PubMed Abstract | Publisher Full Text\n\nEcker DJ, Butt TR, Sternberg EJ, et al.: Yeast metallothionein function in metal ion detoxification. J Biol Chem. 1986; 261(36): 16895–16900. PubMed Abstract\n\nKlaassen CD, Liu J, Choudhuri S: Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol. 1999; 39: 267–94. PubMed Abstract | Publisher Full Text\n\nGoering PL, Klaassen CD: Altered subcellular distribution of cadmium following cadmium pretreatment: possible mechanism of tolerance to cadmium-induced lethality. Toxicol Appl Pharmacol. 1983; 70(2): 195–203. PubMed Abstract | Publisher Full Text\n\nPark JD, Liu Y, Klaassen CD: Protective effect of metallothionein against the toxicity of cadmium and other metals(1). Toxicology. 2001; 163(2–3): 93–100. PubMed Abstract | Publisher Full Text\n\nConrad CC, Walter CA, Richardson A, et al.: Cadmium toxicity and distribution in metallothionein-I and -II deficient transgenic mice. J Toxicol Environ Health. 1997; 52(6): 527–543. PubMed Abstract | Publisher Full Text\n\nBrandão R, Santos FW, Farina M, et al.: Antioxidants and metallothionein levels in mercury-treated mice. Cell Biol Toxicol. 2006; 22(6): 429–438. PubMed Abstract | Publisher Full Text\n\nShariati F, Esaili Sari A, Mashinchian A, et al.: Metallothionein as potential biomarker of cadmium exposure in Persian sturgeon (Acipenser persicus). Biol Trace Elem Res. 2011; 143(1): 281–291. PubMed Abstract | Publisher Full Text\n\nKnapen D, Reynders H, Bervoets L, et al.: Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations. Aquat Toxicol. 2007; 82(3): 163–172. PubMed Abstract | Publisher Full Text\n\nTapiero H, Tew KD: Trace elements in human physiology and pathology: zinc and metallothioneins. Biomed Pharmacother. 2003; 57(9): 399–411. PubMed Abstract | Publisher Full Text\n\nPeroza EA, Schmucki R, Güntert P, et al.: The beta(E)-domain of wheat E(c)-1 metallothionein: a metal-binding domain with a distinctive structure. J Mol Biol. 2009; 387(1): 207–218. PubMed Abstract | Publisher Full Text\n\nCoyle P, Philcox JC, Carey LC, et al.: Metallothionein: the multipurpose protein. Cell Mol Life Sci. 2002; 59(4): 627–47. PubMed Abstract | Publisher Full Text\n\nPalmiter RD: The elusive function of metallothioneins. Proc Natl Acad Sci U S A. 1998; 95(15): 8428–8430. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh RK, Anandhan S, Singh S, et al.: Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses. Protoplasma. 2011; 248(4): 839–847. PubMed Abstract | Publisher Full Text\n\nHuang GY, Wang YS: Expression analysis of type 2 metallothionein gene in mangrove species (Bruguiera gymnorrhiza) under heavy metal stress. Chemosphere. 2009; 77(7): 1026–9. PubMed Abstract | Publisher Full Text\n\nAliakrinskaia IO: Some adaptations of Monodonta turbinata (Born, 1780) (Gastropoda, Prosobranchia, Trochidae) to feeding and habitation in the littoral zone. Izv Akad Nauk Ser Biol. 2010; 37(1): 63–68. Publisher Full Text\n\nAmiard JC, Amiard-Triquet C, Barka S, et al.: Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat Toxicol. 2006; 76(2): 160–202. PubMed Abstract | Publisher Full Text\n\nRoesijadi G: Metallothionein and its role in toxic metal regulation. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1996; 113(2): 117–123. Publisher Full Text\n\nHasan MK, Cheng Y, Kanwar MK, et al.: Responses of plant proteins to heavy metal stress-a review. Front Plant Sci. 2017; 8: 1492. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Y, Yang H, Liu N, et al.: Cadmium accumulation and metallothionein biosynthesis in cadmium-treated freshwater mussel Anodonta woodiana. PLoS One. 2015; 10(2): e0117037. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSakulsak N, Talek K, Sukjai K, et al.: Metallothionein and epidermal growth factor expressions in wild rodent submandibular gland living in cadmium-contaminated area, Mae Sot, Tak by immunohistochemistry staining. The 32nd AAT Annual Conference, Thailand. 2009; (suppl): 53–55.\n\nKavun VY, Shulkin VM, Khristoforova NK, et al.: Metal accumulation in mussels of the Kuril Islands, north-west Pacific Ocean. Mar Environ Res. 2002; 53(3): 219–226. PubMed Abstract | Publisher Full Text\n\nZangger K, Shen G, Oz G, et al.: Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem J. 2001; 359(Pt 2): 353–360. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "43030",
"date": "04 Feb 2019",
"name": "Ima Yudha Perwira",
"expertise": [
"Reviewer Expertise Water polution and ecotoxicology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nThis paper explains about the usage of Metallothionein (MT) expression as a specific biomarker for the presence of cadmium (Cd), mercury (Hg), and lead (Pb) in the tissues (gills and stomach) of oysters (Crassostrea cuculata and Crassostrea glomerata) and the coastal waters of the South coast of East Java. This study has used the appropriate methodologies to measure several parameters. The heavy metals (Pb, Cd, and Hg) are measured using atomic absorption spectrophotometry (AAS). Heavy metals in the water samples are directly measured by using AAS, while that in the gill tissue and stomach are extracted (using nitric acid and incubated at 5-8oC for 30 minutes) before the measurement. The expression of MT is observed by using confocal laser scanning microscopy (CLSM). The authors also mentioned the detailed measurement of water quality for supporting the data of this study. The result of this study showed that the heavy metal content in the water is generally past the specific quality standard (based on the Decree of the State Minister of Environment No. 51 of 2004 concerning seawater quality standards for heavy metal content) (Jelaskan kenapa bisa tinggi). The analysis of MT expression on both gill tissue and stomach showed the highest value in the Prigi station. This study also showed a strong relationship between heavy metals (Pb, Cd, and Hg) with gill tissue and stomach in both Crassostrea cuculata and Crassostrea glomerata.\nComments:\nThis study is interesting, but some points need to be considered. It would be better for the authors to mention some previous study about the using of MT expression as biomarker in the marine species. This study is also interesting because it was carried out in the south coastal area of Java Sea. There is industrial activity located in this area. It would be nice if the authors could mention the importance of doing this study in that area. The highest heavy metal content in the gill tissue and stomach of oysters is observed in the Prigi and Sendang Biru stations. The author has compared the heavy metal content in the oyster with Mytilus and Thylacodes. It would be better if there is another comparative result on the oyster.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "57619",
"date": "29 Jan 2020",
"name": "Rossita Shapawi",
"expertise": [
"Reviewer Expertise Aquaculture",
"Aquaculture feed development",
"fish nutrition",
"applied phycology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle\n\nI would suggest a title that contain \"heavy metal\" term in it. Not everyone is familiar with the word metallothionein. e.g. \"Determination of heavy metal contents of oysters (Crassostrea cuculata and Crassostrea glomerata) from the .....using metallothionein expression method\"\n\nAbstract\nBackground - ...MT level in both types of oyster (replace types with species).\n\nConclusions - The sentence needs to be improved - Eg. The level of heavy metals ...has a strong relationship with MT...\n\nIntroduction\nThis section requires language editing. You also need to mention the purpose of comparing two different species of oysters in this study.\n\nMethods\nHow many samples (n) altogether were used in the study? I cant find any information on this. The first line stated that three samples of oysters were used? Were these three groups of samples?\n\nSeveral typo/formatting issues here to be fixed.\n\nResults and Discussion\nSimilarly, this section also requires language editing.\n\nYou mentioned that your findings in soft tissue exhibit the same pattern with Bilgin et al. - Pb>Cd>Hg??? Figure 2 shows Pb>Hg>Cd ????\n\nThere is no discussion on the background of the sampling areas. Any particular differences among the three areas (Eg. industrial activities) that may influence the results?\n\nI suggest the \"Water quality analysis\" to be presented before other results and discussion.\n\nGeneral\nThe findings of the present study is considered very significant to the local community. I recommend this manuscript to be accepted for publication with minor corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5445",
"date": "30 Apr 2020",
"name": "Asus Hertika",
"role": "Author Response",
"response": "1. InquiryI would suggest a title that contain \"heavy metal\" term in it. Not everyone is familiar with the word metallothionein. e.g. \"Determination of heavy metal contents of oysters (Crassostrea cuculata and Crassostrea glomerata) from the .....using metallothionein expression method\" Response: Thanks for the reviewer's suggestion. We prefer to use this title to reviewer title suggestion. The word “determination of heavy metal….using metallothionein methods” in reviewer suggestion is not proper. Because we can’t determine heavy metal content using metallothionein. However, our title more proper to describe the article.. 2. InquiryBackground - ...MT level in both types of oyster (replace types with species). Conclusions - The sentence needs to be improved - Eg. The level of heavy metals ...has strong relationship with MT... Response: Thank you very much for your concern and suggestion. In the background of abstract, we already replace types with species. Furthermore, in the conclusion of abstract we have changed the sentence. 3. InquiryThis section requires language editing. You also need to mention the purpose of comparing two different species of oysters in this study. Response: The reviewer raises an interesting concern. The purpose of comparing two different species of oysters in this study detail already put. 4. InquiryHow many samples (n) altogether were used in the study? I cant find any information on this. The first line stated that three samples of oysters were used? Were these three groups of samples? Several typo/formatting issues here to be fixed. Response: Thank you very much for your valuable suggestion. We have written the detailed total sample we have fixed several issues or typo. There are three groups sample from different location that the oyster was taken. They are sendang biru (group 1), Prigi Beach (Group 2), Popo beach (group 3). In each group have 3 substation sampling. In this study there are 108 oyster in total was used. 5. InquiryResults and DiscussionSimilarly, this section also requires language editing. You mentioned that your findings in soft tissue exhibit the same pattern with Bilgin et al. - Pb>Cd>Hg??? Figure 2 shows Pb>Hg>Cd ???? Response: the reviewer raises an interesting concern and valuable suggestion. Thanks for the question. In this case, the result showed that the content of Pb was higher than Cd and Hg, however, It still has same result with bilgin et al that Pb has higher content than Cd and Hg. Further, we have made the new sentence of this already. 6. InquiryThere is no discussion on the background of the sampling areas. Any particular differences among the three areas (Eg. industrial activities) that may influence the results?Response: The reviewer raises an interesting concern and valuable suggestion. Each sampling area has difference characters. We have put the detailed the differences among the three areas. 7. InquiryI suggest the \"Water quality analysis\" to be presented before other results and discussion.Response: The reviewer raises an interesting concern. We have put the water quality analysis at the before other results and discussion follow reviewer suggestion."
}
]
}
] | 1
|
https://f1000research.com/articles/8-56
|
https://f1000research.com/articles/9-309/v1
|
29 Apr 20
|
{
"type": "Opinion Article",
"title": "Confronting Covid-19 by exploring the possibility of vaccinating with live SARS-CoV-2 virus itself, via a route that would reduce the incidence of pulmonary complications",
"authors": [
"Etienne Joly"
],
"abstract": "This article proposes that one should explore whether the pulmonary complications of Covid-19 can be reduced or avoided by bypassing the airway entry of the SARS-CoV-2 virus. This could possibly be achieved by injecting live SARS-CoV-2 virus intradermal (ID), subcutaneous, intra-muscular (IM) or intra-peritoneal (IP), or by targeting the virus to the digestive tract. The effectiveness and innocuity of using those various routes could be tested very rapidly in animal models, such as Macaques, Hamsters, Ferrets or Cats.\n\nThe hope is that these experiments will reveal a route of inoculation that can reliably lead to bona-fide infections, resulting in strong immune responses, with both cellular and serological components, but with much less viral replication in the lungs. This would not only hopefully reduce the incidence of pulmonary complications in the infected subjects, but would also probably reduce the amount of virus released by them via aerosols, and thus reduce the vector of contagiosity that is hardest to control, and that probably leads most effectively to viral replication in the lungs.\n\nIf those experiments in animal models reveal that one or several routes can be used effectively to reduce pulmonary pathology, a clinical trial could be conducted in human volunteers with very low risk profiles. The ID route should probably be considered as a priority, since it could double-up as a skin test to reveal the immune status of the recipients towards the SARS-CoV-2 virus.\n\nThe course of action proposed here may possibly provide a way of taking a step ahead of the virus, and if it works as hoped, could help to end the need for confinement within a matter of months, if not weeks.",
"keywords": [
"SARS-CoV-2",
"vaccination",
"clinical trial"
],
"content": "Identifying the real enemy\n\nAs of 21 April, over 2 million cases of Covid-19 have been recorded worldwide and thousands are dying every day (https://ourworldindata.org/coronavirus). It is now clear, however, that the virus only kills a small proportion of those it infects, whilst others recover and become immune in a matter of days. The final outcome of this sanitary war is therefore a foregone conclusion, and the survival of the human race is clearly not at stake.\n\nFrom the above paragraph, it is clear that the true enemy is not the virus, but time. The true question therefore is: how long will it take to resolve the crisis, and at what human and economic costs?\n\nWe thus desperately need to identify rapidly a strategy to reach the only situation that will allow us to put an end to confinement: herd immunity. The approach proposed here consists of exploring if the SARS-CoV-2 virus can be turned into an ally to save time towards reaching suitable levels of herd immunity, and with this the resolution of this unprecedented worldwide crisis.\n\nSARS-CoV-2 is a highly contagious virus that has two major peculiarities (Guan et al., 2020; Zhu et al., 2020):\n\nThe vast majority of serious cases are due to pulmonary complications, following the entry of the virus via the airways and its initial replication in the lungs.\n\nThe morbidity of Covid-19 is massively skewed towards elderly patients, as well as those with pre-existing medical conditions.\n\nIt is now clear that the virus causes only mild symptoms in a very significant proportion of the people it infects, and many can even remain completely asymptomatic (Bendavid et al., 2020). Whilst this is fortunate for those asymptomatic carriers, this is also a major factor that must have contributed to the spread of the virus to every single corner of the inhabited world over the course of just a few weeks.\n\nThe only way to evaluate what proportions of various populations have been infected by SARS-CoV-2 will be to carry out surveys of the immunological status of large numbers of people representative of those populations. Serological tests are by far the simplest way to carry out such surveys, but the results of those tests can be relatively tricky to interpret because of significant levels of false positives, due to cross-reactivity with other coronaviruses, and false negatives, due to delayed rise in antibody titres (Okba et al., 2020), or to a mostly cellular immune response in certain patients (Jin et al., 2020).\n\nAt any rate, the preliminary results of the serological studies carried out on general populations in severely affected areas, where still less than 1% of the population did show clinical signs, seem to suggest that somewhere between 10 and 20% of the population could be already immune to the virus (Regalado, 2020).\n\nSerological testing will identify people who, having already been infected by SARS-CoV-2 and become immune to it, could come out of confinement and resume a normal life without being a danger to themselves or to others. Several countries are considering issuing sanitary passports to such people. Since there are almost no serious cases in people under 40 years of age with no pre-existing medical condition, if the confinements were to continue for much longer, one could envisage situations in which younger people will start demanding the right to return to a normal life, risking infection by the virus, and thus also risking spreading it to high risk individuals they would interact with. The strategy proposed here represents a possible solution.\n\nLong-lasting confinement will be especially punitive in countries where many people have no source of income unless they go to work. For such people, confinement will rapidly result in starvation, and when hunger hits populations, civil unrest is usually not very far behind. Large numbers of Americans, for example, are employed on “at will” contracts, and have no health benefits. Of greater concern, in several African countries, the confinement imposed by some regimes is predicted to result in the starvation of very large numbers of the poorest people over the coming weeks, due to what has been dubbed ‘Pochvid-20’.\n\nIn the strategy proposed here, to speed up the process of developing herd immunity, I suggest taking an approach that would be inconceivable under normal circumstances. Some of the shortcuts suggested here may result in a certain number of casualties. On the other hand, one should not forget that, in the current climate, thousands of people are dying every day, from the virus, in addition to those that are suffering from the consequences of confinement, and that every day saved could thus save as many lives.\n\n\nTechnical aspects\n\nImmunizing with live viruses has long been recognized as the best way to obtain long-lasting immunity, and the skin is well known as a prime site for immunization, yielding responses that are generally skewed towards a type I T helper response (Zehrung & Kristensen, 2009). A strong, long-lasting cellular immunity raised against the virus itself rather than against a recombinant construct may be the best way to avoid the manifold problems raised by antibody dependent enhancement (ADE) (Kulkarni, 2020; Ricke & Malone, 2020; Smatti et al., 2018). ADE is one of the major problems that many vaccines developed against lung pathogens have repeatedly encountered over the years. This is one of the reasons why, almost 20 years after the outbreak, we still do not have a vaccine available against SARS-CoV-1, and why a recombinant vaccine against SARS-CoV-2 cannot reasonably be expected to be ready for broad use sooner than 18 months, at the very least (Ricke & Malone, 2020). On the other hand, the live SARS-CoV-2 virus is the ‘real thing’ and it will have infected well over 2 million people worldwide before the end of this month. Its effects will have been documented better and faster than any other virus before in history, and it is clear by now that lung complications are the primary problem caused by this new virus.\n\nAttenuated viruses have been used to develop very effective vaccines against many of the most lethal viruses, including rabies, yellow fever, polio, measles, mumps, rubella and chicken pox. Some of these vaccines have been found to work after ID inoculation (Hickling et al., 2011; Zehrung & Kristensen, 2009). But obtaining those attenuated viruses took many years of passaging in animals, or in cultured cells, and subsequent testing took at least as long to ensure that the attenuated viruses resulted in suitable immune memory in vaccinated human subjects.\n\nWith Covid-19, we do not have this sort of time available, and I would argue that, in the low risk population, the SARS-CoV-2 virus already has the low level of pathogenicity that would make it potentially suitable for a candidate vaccine. Therefore, what I propose to explore is if, by simply changing its route of entry into the organism, one could not turn it into a virus with sufficiently low pathogenicity to be suitable for mass vaccination.\n\nMacaques have already been found to represent a suitable animal model for Covid-19 (Bao et al., 2020; Rockx et al., 2020); recent evidence reports that cats and ferrets (Shi et al., 2020) or hamsters (Chan et al., 2020) could also be used, which would open the way for exploring many more avenues in parallel, provided that suitable facilities for performing experiments on those animals can be identified. On the subject of the inoculation route influencing the distribution of the virus, one should highlight that, in a preliminary study, Deng et al. found that one of the two macaques inoculated via the eye’s conjunctival route harboured different tissue distributions of viral loads compared to the one macaque inoculated intratracheally (Deng et al., 2020). On this subject, it would be of interest to explore to what extent the different possible routes of contagion by SARS-CoV-2 may contribute to the astounding variability in the intensity of Covid-19 between different patients of similar profiles. Could it be, for example, that lung contagion through aerosols tends to result in more serious clinical outcomes than if the virus is swallowed after passing from hand to mouth?\n\nTo bypass the airways, I will argue below that the ID route should be explored as a priority, in one or several of the animal models available if resources permitted it. If the ID route does not result in the efficient triggering of systemic infections, leading to strong immunity and rapid recovery, one could also envisage using subcutaneous, intra-muscular or intra-peritoneal injections, as well as digestive routes, either by ingestion of enterically coated formulations, or via simple intra-rectal (IR) inoculation. If resources permitted, a preferable course of action to save time would be to explore as many of these routes in parallel as possible.\n\nThe ID route would, however, seem preferable to the other routes of inoculation, for the following reasons:\n\nThe ID route could also double-up as a skin test, akin to the Mantoux test used to test for BCG-primed immunity. Thus, individuals previously infected by SARS-CoV-2 could be detected in a matter of days. These individuals could then be issued with a ‘sero-converted’ sanitary passport and resume a normal life (and contribute to running the health care systems and to restarting the countries’ economies, without needing to use the protection equipment which is in short supply in so many places).\n\nIt could also boost the immunity of those who have only had mild forms of Covid-19. These people would possibly be at high risk of ADE-related complications in the form of a cytokine storm if they were to be accidentally re-infected by SARS-CoV-2 via the airway route in the course of later months or years, when their level of circulating antibodies would have decreased to sub-neutralizing levels (Ricke & Malone, 2020; Smatti et al., 2018).\n\nA further advantage of the ID route is that vaccination could be carried out with innocuous material such as micro-needles arrayed on stamps rather than syringes and needles (Kim et al., 2020). And since the virus would only be inoculated into the superficial strata of the skin, the necessary sanitary standards may be more easily attained than for materials destined to be injected with regular needles. Efforts of mass vaccination would therefore be easier to implement.\n\nTo conclude, an advantage of the ID route in my eyes is that with the use of the stamp-like devices carrying micro-needles, volunteers in the early phase trials could self-inoculate the virus, which could circumvent some of the serious ethical issues raised by the approach proposed here.\n\nIR inoculation may turn out to be an alternative to the ID. Indeed, for the IR route, the sanitary considerations would be minimal, and simple dilutions of viral preparations derived directly from tissue culture could conceivably be used. As experience with HIV has shown, some viruses can spread very effectively via the rectal route. Given the fact that ACE-2, the viral receptor, is expressed at very high levels in the digestive tract (see https://www.proteinatlas.org/ENSG00000130234-ACE2/tissue), the virus is expected to replicate efficiently in the gut, and consequently to promote strong mucosal immunity. This is supported by the observation that digestive signs are indeed frequent in Covid-19 patients (Pan et al., 2020), but they do not seem to lead to life threatening complications as frequently as in the lungs (https://www.sciencemag.org/news/2020/04/how-does-coronavirus-kill-clinicians-trace-ferocious-rampage-through-body-brain-toes#). As an elaboration, one could also conceive of using the IR route as an initial primer, followed by ID inoculation 7 to 10 days later, which would not only boost the immunity, but also provide a direct and sensitive test of the immune status of the recipients.\n\nIf the ID or IR routes of inoculation of live SARS-CoV-2 virus were found to cause complications too frequently in the initial stages of the experimentation, a way to work around this undesirable situation may be to use an approach inspired from that used for Polio vaccination nowadays, i.e. the combination of a recombinant live or inactivated vaccine as a first step, followed by inoculation with live virus. Indeed, two different vaccines were initially developed and used against Polio: the inactivated Salk vaccine, which has limited efficacy, but does not have any serious side effects, and the Sabin vaccine, which is using a live attenuated vaccine (the one that is given to kids on a lump of sugar), and provides lifelong immunity. The major drawback of this live vaccine is that it has been found that the attenuated polio virus rapidly reverts to a wild-type, fully pathogenic virus in the intestine of the vaccinated subjects, who will then expel it in very large numbers in their faeces. This is only a very infrequent problem for the vaccinated subjects themselves (in 1/750,000 cases)(John & Vashishtha, 2013), but is of much more concern for the surrounding family members if they are not vaccinated. Nowadays, the recommended course of action for polio vaccination comprises first an immunisation with the inactivated virus, followed a few weeks later by immunisation with the live attenuated virus.\n\nThe logistics of such a two-step approach for fighting Covid-19 would, however, be more complicated than the simple inoculation of live virus in a single step. It has been found that the SARS-CoV-2 can easily be grown to very high titres (107/ml) in cell lines such as Vero, Caco II, or Calu 3 cells (Hoffmann et al., 2020). With just a few tens of litres of infected cultured cells, one would thus expect to have generated enough live virus to inoculate the whole of the world’s population. But the amount of viral material could become a limiting factor if a pre-immunisation step had to be carried out. Indeed, even if, accounting for the experience acquired with other intra-dermally applied vaccines, another advantage of the ID route is that it would require less material for the inoculation than other routes, such as subcutaneous or intramuscular (Hickling et al., 2011), immunisation with inactivated virus would require several orders of magnitude more material, and thus much more time to produce, than if just live virus was used. And one of the major disadvantages of using inactivated or recombinant vaccines is that, after the vaccination of the first cohort of volunteers, one has to wait to see if the vaccine is indeed protective, which can take a very long time if one waits for the infections to occur naturally. Others have argued that, in order to save time, one should consider inoculating those volunteers with the virus after vaccination (Eyal et al., 2020); this may, however, raise even more ethical issues than the approach proposed herein, especially if one considers the frequent problems of ADE encountered with vaccines against viruses that can cause lung pathologies (Ricke & Malone, 2020). Personally, given the choice between receiving a live virus via the aerial route after vaccination with a new vaccine or via a route which has been found to seldom lead to lung pathology, I would not hesitate to choose the latter.\n\nThe SARS-CoV-2 virus is relatively innocuous for a very large proportion of the population. But a major concern regarding the evolution of this pandemic is that the virus could mutate to become more pathogenic. A further advantage of an approach based on inoculating the virus is that this would completely block any uncontrolled evolution of the virus, as may happen if it is left to spread through populations by simply lifting confinement. It would also mean that serum obtained from any of the immunised recipients could be used to treat those volunteers that would show complications, without any concern for the fact that the virus may be of a sufficiently divergent source that the transferred antibodies would not neutralise the virus efficiently (Jawhara, 2020).\n\n\nThe anticipated long-run scenario(s)\n\nThe hope is that initial experiments carried out on one or several of the available animal models would find that inoculations with live virus via the ID, IR, or some other route will result in the efficient triggering of systemic infections with SARS-CoV-2 with less pulmonary distribution of the virus than the airway inoculation, and hopefully in faster resolution. If this is indeed found to be the case, one could then envisage to setup a first cohort of a few dozens of human volunteers for inoculation with live virus, via the route(s) that will have been identified as the most promising through animal experimentation. Those volunteers would be selected from populations with very low risks of complications. The trial would also have to take place in a completely confined environment, and under close medical monitoring. I suggest that a military base would be a particularly appropriate environment for this type of project.\n\nFurthermore, it does not seem unreasonable to hope that, by then, some therapeutic approaches will have been identified to combat the virus in patients presenting complications. In this regard, passive immunisation with serum from recovered patients seems to be one of the more hopeful avenues to date (Jawhara, 2020; Xie et al., 2020). In the event that one or more volunteers would show signs of serious complications, it is therefore to be hoped that well-defined therapeutic approaches would be at hand by then to prevent truly serious complications from developing in those volunteers.\n\nIn an ideal scenario, the course could be as follows:\n\n1) Over the coming month, completion of the ID inoculation experiments in macaques (with hamsters, ferrets and/or cats as potential additions).\n\n2) During this time, preparations will be made for the first trial on a cohort of a few dozens of volunteers in a completely confined environment. This could take place during the coming month(s), but will obviously only go ahead if and when the results of the above experiments will have shown very promising results.\n\n3) If and when the project gets to that stage of self-inoculating by human volunteers, one of the very important aspects of this experimental stage will be to determine how contagious these inoculated volunteers will become, especially whether they excrete a lot of live infectious virus through their airways, urine, or faeces.\n\n4) If it is found that, after inoculation, the volunteers are not worryingly contagious, one could conceive of an exercise of mass immunisation, strictly on volunteers, asking the inoculated volunteers to stay confined at home during the time they generate immunity to SARS-CoV-2.\n\n5) On the other hand, if it turned out that inoculated individuals can become highly contagious, one may have to resort to performing the immunisations in confined centres, which I refer to as ‘covidodromes’. For this, I envisage that military bases could provide convenient environments, at least for the first stages of the exercise.\n\n6) If it turned out that the approach proposed here was truly efficient, and could result in the rapid sero-conversion of thousands of people with a bare minimum of complications, and even fewer casualties, one could then endeavour to step-up the process to be in a position to offer covidodromes for millions of volunteers to travel to and be inoculated with controlled stocks of virus, in a controlled environment. This could for example be achieved by using tourist places which are currently deserted, such as holiday centres and villages, ski resorts, or even some of the large islands such as those found along the French littoral.\n\nWith such a large-scale approach, significant proportions of sero-converted people in the population could be achieved before this summer, ultimately reaching herd immunity, which is the only thing that will allow us to come out of confinement serenely.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "Acknowledgements\n\nI am employed by INSERM. I am grateful to Tom McKee for suggesting that the digestive route should be explored in parallel to others, and to Denis Hudrisier for his feedback on an earlier stage of the manuscript.\n\n\nReferences\n\nBao L, Deng W, Gao H, et al.: Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv. 2020. Publisher Full Text\n\nBendavid E, Mulaney B, Sood N, et al.: COVID-19 Antibody Seroprevalence in Santa Clara County, California. medRxiv. 2020. Publisher Full Text\n\nChan JF, Zhang AJ, Yuan S, et al.: Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis. 2020; pii: ciaa325. PubMed Abstract | Publisher Full Text\n\nDeng W, Bao L, Gao H, et al.: Rhesus macaques can be effectively infected with SARS-CoV-2 via ocular conjunctival route. bioRxiv. 2020. Publisher Full Text\n\nEyal N, Lipsitch M, Smith PG: Human challenge studies to accelerate coronavirus vaccine licensure. J Infect Dis. 2020; pii: jiaa152. PubMed Abstract | Publisher Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med. 2020. Publisher Full Text\n\nHickling JK, Jones KR, Friede M, et al.: Intradermal delivery of vaccines: potential benefits and current challenges. Bulletin of the World Health Organization. 2011; 89(3): 221–226. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffmann M, Kleine-Weber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2): 271–280.e8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJawhara S: Could Intravenous Immunoglobulin Collected from Recovered Coronavirus Patients Protect against COVID-19 and Strengthen the Immune System of New Patients? Int J Mol Sci. 2020; 21(7): pii: E2272. PubMed Abstract | Publisher Full Text\n\nJin Y, Wang M, Zuo Z, et al.: Diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019. Int J Infect Dis. 2020; 94: 49–52. PubMed Abstract | Publisher Full Text\n\nJohn TJ, Vashishtha VM: Eradicating poliomyelitis: India’s journey from hyperendemic to polio-free status. Indian J Med Res. 2013; 137(5): 881–894. PubMed Abstract | Free Full Text\n\nKim E, Erdos G, Huang S, et al.: Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development. EBioMedicine. 2020; 102743. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKulkarni R: Antibody-Dependent Enhancement of Viral Infections. In: Bramhachari, P. V. ed. Dynamics of immune activation in viral diseases. Singapore: Springer Singapore, 2020; 9–41. Publisher Full Text | Free Full Text\n\nOkba NMA, Müller MA, Li W, et al.: Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerg Infect Dis. 2020; 26(7). PubMed Abstract | Publisher Full Text\n\nPan L, Mu M, Yang P, et al.: Clinical Characteristics of COVID-19 Patients With Digestive Symptoms in Hubei, China: A Descriptive, Cross-Sectional, Multicenter Study. Am J Gastroenterol. 2020. PubMed Abstract | Publisher Full Text\n\nRegalado A: Blood tests show 14% of people are now immune to covid-19 in one town in Germany. 2020. Reference Source\n\nRicke DO, Malone RW: Medical Countermeasures Analysis of 2019-nCoV and Vaccine Risks for Antibody-Dependent Enhancement (ADE). 2020. Publisher Full Text\n\nRockx B, Kuiken T, Herfst S, et al.: Comparative Pathogenesis Of COVID-19, MERS And SARS In A Non-Human Primate Model. bioRxiv. 2020. Publisher Full Text\n\nShi J, Wen Z, Zhong G, et al.: Susceptibility of ferrets, cats, dogs, and different domestic animals to SARS-coronavirus-2. Science. 2020; Apr 8. pii: eabb7015. Publisher Full Text\n\nSmatti MK, Al Thani AA, Yassine HM: Viral-Induced Enhanced Disease Illness. Front Microbiol. 2018; 9: 2991. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXie Y, Cao S, Li Q, et al.: Effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with COVID-19. J Infect. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZehrung D, Kristensen D: Intradermal Delivery of Vaccines: A review of the literature and the potential for development for use in low- and middle- income countries. WHO. 2009. Reference Source\n\nZhu N, Zhang D, Wang W, et al.: A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "68210",
"date": "29 Jul 2020",
"name": "Ji‐Ming Chen",
"expertise": [
"Reviewer Expertise public health",
"veterinary medicine",
"control of infectious diseases",
"epidemiology",
"influenza",
"virology",
"evolution"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 to use a wild-type SARS-CoV-2 as a live vaccine through intradermal, subcutaneous, intra-muscular, or intraperitoneal inoculation, to bypass the pathogenesis site of the fragile lungs. I published three articles discussing the same opinion (PMID: 323200831; PMID: 325255872; PMID: 326816503). This opinion is supported by the fact that two wild-type adenoviruses have been SAFELY used as live vaccines through enterically coated capsules for 40 years in the US recruits, and reduced over 99% cases of adenovirus-associated acute respiratory diseases (PMID: 274754744). My own experiments also suggested that intra-muscular inoculation of a wild-type respiratory virus is safe and able to induce strong immunity (PMID: 326816503).\n\nWith my own experiment, I do believe that it is POSSIBLE and ONLY possible that intradermal, subcutaneous, or intra-muscular inoculation of SARS-CoV-2 is safe and able to induce strong immunity. We should use animal experiments to confirm this route, and such animal experiments are not expensive as compared with the fact that so many people are dying every day and so much economic loss is happening worldwide.\nOn the other side, some methods available readily could enhance the safety of the live unattenuated vaccine of COVID-19. For instance, if the SARS-CoV-2 is produced using PK-15 cells rather than Vero cells, it shall carry α1,3-galactose (αGal), and thus can form immune complexes with anti-αGal antibodies which are naturally abundant in human bodies. The anti-αGal antibodies could greatly enhance the safety of the live unattenuated vaccine through blocking the viral entry, aiding complements to destroy the virus, and aiding immune cells to destroy the virus (PMID: 326816503). Meanwhile, the anti-αGal antibodies could greatly enhance the efficacy of the live unattenuated vaccine through immune opsonization, as the immune complexes aid the antigen capture, process and presentation of APCs (PMID: 326816503). Again, this view should be investigated using animal experiments.\nIf the above animal experiments strongly support the opinions, the live unattenuated vaccine of COVID-19 should enter clinical trials in the volunteers.\n\nFrom the view of a veterinary scientist and a long-term worker in public health, I do believe that the live unattenuated vaccine could be much more powerful than the dozens of vaccines currently under rapid development for COVID-19, because the unattenuated vaccine could be of high development speed, high production efficiency, ability to be produced in more countries, all of which could be as important as the safety of vaccine for reducing deaths and losses of the pandemic.\nIn short, I fully support the major opinions of this view: (1) the unattenuated vaccine could be powerful to curb the COVID-19 pandemic; (2) the unattenuated vaccine is imperatively desired to be investigated using animal experiments. Meanwhile, the author should consider the relevant history, publications, and methods to enhance the safety and efficacy of the unattenuated vaccine.\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": []
},
{
"id": "83933",
"date": "05 May 2021",
"name": "Ravi Kant Narayan",
"expertise": [
"Reviewer Expertise Anatomy",
"Genetics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article proposed the use of unattenuated wild-type SARS-CoV-2 as a live vaccine to be inoculated through routes that can bypass the lungs. Taking into purview the recent trends of the COVID-19 pandemic, the opinion seems to be justified. If we look at the present scenario of the pandemic in India, it would be true to say that the world is going to witness nature's wrath due to human complacency. To avoid further deterioration outside-the-box thinking is required, this article presents the very same.\n\nRecent reports have presented facts that differ from those stated in this article. It mentions \"Since there are almost no serious cases in people under 40 years of age with no pre-existing medical condition\", this can be now refuted by Abbasi (2020)1, which mentions the changing scenario of the pandemic with relation to age distribution.\nTo conclude, I would suggest that the opinion being presented should be experimented with in order to search all the avenues.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "66390",
"date": "10 May 2021",
"name": "Jonathan Yewdell",
"expertise": [
"Reviewer Expertise Adaptive immunity to viruses"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nJoly proposes immunization with live SARS-CoV-2 (CoV2) via non-respiratory routes as a rapidly employable and cost-effective vaccine. This is a brilliant proposal but compromised somewhat by the introduction of highly effective vaccines for COVID-19. I would recommend that the author update the article accordingly and possibly alter the message to incorporate:\nCoV2 vaccine effectiveness.\n\nAdvantages of GI immunization for possibly optimizing mucosal immunity. Possibly mention existing enteric adenovirus vaccines used for US military personnel as a relevant example.\n\nMake the obvious point that the proposed approach could be rapidly deployed for emerging CoV2 variants or future pandemic with zoonotic virus.\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": "76622",
"date": "24 May 2021",
"name": "Mahmoud Kandeel",
"expertise": [
"Reviewer Expertise Pharmacology"
],
"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 concept of using life viruses by injection in atypical location might be attractive to some extent and some other researchers had already published on it. However, I am still not encouraged to support this premise as long as safer and effective techniques can be developed. Recently, several vaccines have been approved and widely practiced worldwide, the opinion must be updated.\nThe application of live viruses for injection of human being can be a matter of virus activation, mutation and infected or immunized persons might be seeding the virus in the surrounding community and act as a source of infection. This application requires also much care, sophisticated resources and extreme care from the medical staff to avoid contact with the live virus.\nDespite there are some agreements in the literature, there is no solid evidence for the success of this concept. This manuscript also needs to be updated with the recent findings and approvals in the field of COVID-19 vaccines.\nSince the strength of immunity might depend on the amount of virus proteins and poster doses. I am not sure that small amount of live SARS-CoV-2 virus are sufficient to give solid immunity.\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? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-309
|
https://f1000research.com/articles/8-1921/v1
|
14 Nov 19
|
{
"type": "Research Article",
"title": "Predictive statistical model for the factors associated with prenatal depression among pregnant adolescents in Maha Sarakham province, Thailand",
"authors": [
"Bhanwasa Jantasin",
"Witaya Yoosook",
"Supawadee Thaewpia",
"Witaya Yoosook",
"Supawadee Thaewpia"
],
"abstract": "Background: The rate of adolescent pregnancy has steadily increased in Thailand over the years and depression is considered an important psychological problem in many pregnant adolescents. The aims of this study were to determine the proportion of pregnant adolescents with depressive symptoms and to identify a predictive statistical model for the factors related to prenatal depression among adolescents at antenatal care clinics in 10 community health centers in Maha Sarakham Province. Methods: A cross-sectional study was conducted in 230 randomly selected participants who were pregnant adolescents who volunteered to participate in the research, those aged ≤19 years, and had an estimated gestational age of ≤18 weeks. Those with severe obstetrics complications or a psychiatric disorder were excluded. The subjects were asked to complete a demographic questionnaire and the Thai version of the Edinburgh Postnatal Depression Scale. The mean age of participants was 17.0 (±1.4) years. Results: Overall, 43.9% of the adolescents had a cut off score of ≥11, suggestive of prenatal depressive symptoms. Multivariate analysis identified that pregnant adolescents at a primary school education level were 6.5 times more likely to experience depression symptoms than those with a diploma. Those who were emotionally supported by their husband or partner compared to relatives had a five times higher chance of presenting depressive symptoms, and those with a perceived bad relationship with their husband or partner seven times higher. The model was well-calibrated (goodness-of-fit test, p = 0.675) and had high discriminative power (ROC curve = 0.73 (95%CI = 0.67 to 0.79). Conclusions: More attention is needed to reduce the known problems with pregnant adolescents and to ensure they continue with their education. Lastly, public education to support mental health initiatives for individuals and families should be encouraged.",
"keywords": [
"Adolescent pregnancy",
"Community hospital",
"Prenatal depression",
"Thai Edinburgh Postnatal Depression Scale"
],
"content": "Introduction\n\nAdolescents can develop health problems during pregnancy that adversely affect both physical and psychological health (UNFPA, 2014b). Pregnant adolescents commonly experience depressive symptoms (Lancaster et al., 2010); the prevalence has been estimated to be approximately 20% (Abajobir et al., 2016; Nasreen et al., 2011; Roomruangwong & Epperson, 2011). Pregnant adolescents with depression and their infants are at a higher risk of increased morbidity (Tzilos et al., 2012), resulting in a greater risk for preterm labor and intrauterine fetal growth retardation (Field, 2017; Grote et al., 2010; Orr et al., 2007). Depressed pregnant adolescents also have a greater risk of suicidal ideations and suicide attempts than those without depression (Hodgkinson et al., 2010).\n\nRisk factors predisposing adult pregnant women to depressive symptoms include daily maternal stress, loss of self-confidence, and inadequate social support and emotional coping (Da Costa et al., 2000; Dennis et al., 2007). Risks factors for depression among pregnant adolescents include not only those listed for adult women but also include unplanned pregnancy, low education, non-marital status, alcohol and drug use, inadequate income, and a history of emotion, sexual, and physical abuse (Koniak-Griffin et al., 1996; Meltzer-Brody et al., 2013; Siegel & Brandon, 2014; Uthaipaisanwong et al., 2015). Because prenatal depression in adolescence is a significant factor for predicting postpartum depression (Dennis et al., 2007), the ability to identify prenatal depression at the earliest stage of pregnancy is particularly important for the health and well-being of mothers and infants and for public health in general.\n\nThailand has a high rate of adolescent pregnancy (Ministry of Public Health, 2017). Studies by Limlomwongse & Liabsuetrakul (2006), and Uthaipaisanwong et al. (2015) in Bangkok and the South of Thailand have shown that the incidence of prenatal depression among adolescents is 46% and 20.5%, respectively. Little research, however, has been conducted in northeastern Thailand, especially in Maha Sarakham province, where the rate of adolescent pregnancy is reported to be high. Therefore, the aim of this study was to estimate the proportion of prenatal depressive symptoms among adolescents in the Maha Sarakham Province, Thailand, and to identify factors associated with prenatal depression symptoms in adolescents.\n\n\nMethods\n\nThis cross-sectional study was conducted from July to October 2017 with 230 pregnant adolescents. The participants were selected using simple randomization in order to avoid sampling bias. Every week, two to three pregnant adolescents were recruited from each antenatal clinic in 10 community hospitals in the Maha Sarakham province, Thailand by randomizing HN numbers (patient identification numbers were drawn as lots) among all of the pregnant adolescents who met the inclusion criteria. Inclusion criteria were 1) pregnant adolescents who volunteered to participate, 2) those ≤19 years of age, 3) and those with an estimated gestational age of ≤18 weeks. The exclusion criteria were pregnant adolescents with obstetric complications or those who had been diagnosed with a psychiatric disorder. Initially, a sample size of 209 adolescents was calculated (Hsieh et al., 1998) assuming a prenatal depression prevalence of 46% (Uthaipaisanwong et al., 2015) with an alpha error of 0.05 and a power of 80%. An extra 21 adolescents (10%) were added to compensate for possible incomplete data, giving a total sample of 230 participants. All participants were recruited in person during their first appointment at the antenatal clinic. After receiving informed consent, participants provided their demographic and socioeconomic information on a written questionnaire (available as Extended data; Jantasin et al., 2019b, Jantasin et al., 2019c) then completed the Thai version of the Edinburgh Postnatal Depression Scale (Pitanupong et al., 2007).\n\nThe Edinburgh Postnatal Depression Scale (EPDS) is an assessment tool available in Thailand to screen for depressive symptoms. Originally developed to detect possible depressive symptoms in the postpartum period (Cox et al., 1987), the EPDS has been successfully used to identify depressive symptons in prenatal women (Eberhard-Gran et al., 2001; Lee et al., 2004). In 2003, the EPDS was translated to Thai by Pitanupong et al. (2007). Three doctorally prepared Thai experts in midwifery reviewed the translation for content validity and approved all items without recommendations. Published psychometric testing supports the 10-item self- assessment questionnaire’s validity and reliability among Thai pregnant women (Pitanupong et al., 2007; Vacharaporn et al., 2003). Preliminary testing for internal reliability using 30 people, not part of the study, showed a Cronbach’s alpha = 0.76.\n\nEach of the ten EPDS items has four response options ranging from 0 to 3. The response descriptors vary somewhat depending on the item’s wording. The participants choose the descriptor most closely indicating their emotions during the prior seven days. Seven items require reverse scoring so that higher total scores are more suggestive of depressive symptoms. A total score can range between 0 and 30. The cutoff score for the EPDS that indicates the presence of depression symptoms has been reported between 10 and 14 (Bolton et al., 1998; Evans et al., 2001). We defined an EPDS score of 11 or greater as a positive screen for depressive symptoms (Uthaipaisanwong et al., 2015).\n\nData analysis were conducted with descriptive statistics to present demographic data. Multivariable analysis through unconditional multiple logistic regression was used to identify factors associated with prenatal depression among pregnant adolescents and to determine potential associations of socio-demographic information with prenatal depressive symptoms (EPDS ≥11=1 and <11=0). Crude analysis through simple logistic regression analysis was used. After that, all variables with p-values less than 0.25 (Hosmer & Lemeshow, 2000) were selected to include an initial regression model for the assessment of multicollinearity. Then, backward elimination for a fitted regression model was performed. The model was developed and its goodness-of-fit was evaluated by determining the predictive accuracy and cut-off that was suitable for clustering towards depressive symptoms among pregnant adolescents using a receiver operating characteristics (ROC) curve. Statistical significance was set at p<0.05. Analyses were done with Statistical Package for the Social Sciences (SPSS) software, version 18.0 (SPSS®, Chicago, USA).\n\nThe study was approved by the Institutional Ethics Committee at Mahasarakham University, Thailand. (Ref No. 054/2560; 21 July 2017). Adolescents of legal age offered their informed consent volunteering to participate in the study. Parents of adolescents younger than 18 years of age provided written informed consent prior to their child’s participation.\n\n\nResults\n\nOf the 230 participants, 101 adolescents (43.9%) met the EPDS cutoff score of ≥11 suggestive of prenatal depressive symptoms. The remaining 129 adolescents (56.1%) scored below the cutoff score (Table 1).\n\nThe mean age of the 101 adolescents was 17.0 (SD±1.4) years old. The majority of those were >15 years old (n=80, 80.0%) and 21 (20.0%) were ≤15 of age. In bivariate analysis, depressive symptoms were significantly correlated with receiving less education, being a single mother with one supporter, having a husband as a supporter, and having a poor relationship with a husband, their family and friends. Pregnant adolescents who only finished primary school were 7.3 times more likely to have prenatal depressive symptoms than those with education to diploma level. In terms of marital status, the adolescents who were single mothers were at 9.4 times higher risk of depressive symptoms than those living together with their partner, with or without being married. Pregnant adolescents who had one supporter were 2.8 times more likely to be at risk of depressive symptoms compared to those with three supporters. The adolescents who had a husband as a supporter tended to be 3.7 times higher at risk of depressive symptoms when compared to those who were supported by relatives. Moreover, pregnant adolescents who reported a bad relationship with their husband, family, and friends were 5.5 times, 2.2 times, 2.8 times higher at risk of depressive symptoms than those with good relationship, respectively (Table 1). Responses to each question from each participant are available as Underlying data (Jantasin et al., 2019a).\n\nAccording to Table 1, eight variables with a p-value of less than 0.25 were selected and included in the initial regression model. From the multiple logistic regression analysis, there were only three factors that were significantly associated with prenatal depressive symptoms: level of education, the perceived support from a husband or partner, and the perceived relationship with a husband or partner (Table 2). Those pregnant adolescents with a primary school education level were 6.5 times more likely to develop prenatal depressive symptoms compared to those with a diploma degree [AOR=6.5 (95% CI: 1.5 to 28.7)]. The pregnant adolescents who perceived support from their husband or partner were five times more likely to have prenatal depressive symptoms compared to those with relatives’ support [AOR=5.1 (95% CI:1.9 to14.5)]. The chance of developing prenatal depressive symptoms in those with a bad relationship with the husband was approximately seven times higher than pregnant adolescents who reported a good relationship [AOR=7.4 (95% CI: 3.1 to17.8)].\n\nThree variables in the final model influencing with prenatal depressive symptoms significantly were as follows:\n\nP(Y=1/X) = 1/[1+EXP(-1.99+ 1.88 Education; Primary + 0.85 Education; Secondary + 1.65 Supporter; Husband/partner + 0.45 Supporter; Their family + 2.00 Relationship with a husband/partner]\n\nTo validate how well the model fits a set of observations, Hosmer-Lemeshow’s goodness-of-fit test was used and the results showed that the model was well-fit data (p-value = 0.675). The results of testing the model's predictive power by creating the relational graph of sensitivity and 1-specificity showed that the model had a good predictive power with ROC of 0.73 (95% CI 0.67 to 0.79) (Figure 1) at the cut point of -0.11, a sensitivity of 57.43, a specificity of 81.40, and an accuracy of 71.30.\n\nArea under ROC curve = 0.73 (95%CI 0.67 to 0.79).\n\n\nDiscussion\n\nThere was an unexpectedly high proportion of pregnant adolescents with prenatal depression symptoms in the ten community hospitals in the Maha Sarakham Province, at 43.9%. This proportion is twice the number reported worldwide (Abajobir et al., 2016; Nasreen et al., 2011; Roomruangwong & Epperson, 2011). However, the findings are similar to the studies reported by Uthaipaisanwong et al. (2015) and Nirattharadorn et al. (2005), with the risk for prenatal depression from EPDS screening at 46% and 47.1%, respectively. Findings indicate that the underlying reasons are associated with the adolescents’ low level of education, the husband or partner who supports her, and the relationship she has with him. Bunevicius et al. (2009) and Lydsdottir et al. (2014) also found that pregnant adolescents with a low level of education are more at risk of having depressive symptoms (see also Field (2017) and Osok (2016)). In Thailand, pregnancy in adolescents often leads to dropping out from school. Consequently, a low level of education limits the opportunities for better employment and earning a higher income, which results in increased stress levels of these pregnant adolescents (Kaewjanta, 2012).\n\nWe found that not only was the emotional support coming from a husband or partner (compared to the pregnant adolescent’s family) a risk factor for depressive symptoms, but also a poor relationship with that person contributes to her depression. This finding seemingly contradicts Stapleton et al. (2012) who found that a husband’s support reduces stress and positively affects pregnant women emotions. However, the perceived emotional support experienced by a pregnant adolescent from her husband or partner needs to be interpreted simultaneously with the finding of the poor relationship she experiences with him. The perceived adverse support from a husband or partner and the negative relationship with him likely increases the adolescents’ depressive symptoms.\n\nFrom the predictive model of prenatal depression symptoms by determining ROC, it was found that level of education, the perceived support from a husband or partner, and the perceived relationship with a husband or partner could predict prenatal depression symptoms well.\n\nThe 95% CI of the narrow ROC reflects that the model had predictive accuracy. Although the sensitivity was not adequate, specificity was at a high level. The model of three variables could be applied in operational planning to prevent and solve the problem of pregnant adolescents with prenatal depression symptoms.\n\nThe current Thai cultural norm is not to provide support to adolescents who become pregnant at a young age; society does not consider them to be ready for motherhood or a marital relationship. Adolescents who become pregnant may prefer to hide their condition from the family. Some may need to leave or transfer to a new school. They may lose their job and income as a result of the pregnancy or because of the change in living arrangements. Frequently, adolescent couples lack the maturity and emotional intelligence to begin a marital relationship, which might lead to domestic violence, although the latter was not part of the study’s investigation or findings. In more agreement with our findings are the reports (Muangpin et al., 2010; UNFPA, 2014a) that pregnant adolescents with depressive symptoms and other mental health problems are adversely affected by the lack of emotional support and a negative relationship with a husband.\n\nThis cross-sectional study does not permit a closer understanding of the long-term trajectory of adolescents who become pregnant and develop depressive symptoms, nor can it examine how their pregnancy might affect their adult lives. This information could come from longitudinal and qualitative studies. In addition, the sample in this study was confined to only one region of the country; therefore, the findings may not be generalizable to other parts of Thailand. Future research might be conducted in other areas, which should consider adult women who became pregnant in adolescence and have shown depressive symptoms and what meaning that held for them in their later lives. Similarly, the husbands or partners of the adolescents should be studied as to their perceptions of the experiences related to unexpected fatherhood with adolescent women.\n\nIn conclusion, the adolescent pregnancy rate in Thailand is increasing and adolescents are vulnerable to suffer from depressive symptoms during pregnancy. As a matter of public health, more attention is needed to help reduce known problems with pregnant adolescents. Efforts to keep adolescents in school or ensure they return to school after delivery of the newborn may be an important step in improving the mental health of adolescent mothers. Public advocacy and education to support mental health initiatives for individuals and couples should be encouraged as a matter of health policy. If couples have become isolated from their families, attempts to reconcile and reunite them by public health personnel may promote better mental health.\n\n\nData availability\n\nFigshare: Raw data - factors associated with depression. http://doi.org/10.6084/m9.figshare.10072898.v2 (Jantasin et al., 2019a).\n\nThis project contains the following underlying data:\n\nRaw-data-factors-associated-with-depression-edit.csv (raw data for each participant extracted in this study).\n\nRaw data Dictionary.docx (data dictionary defining all abbreviations and values)\n\nFigshare: Demographic questionnaire and EPDS in English. http://doi.org/10.6084/m9.figshare.10073012 (Jantasin et al., 2019b).\n\nFigshare: Demographic questionnaire and EPDS in Thai. http://doi.org/10.6084/m9.figshare.10073009 (Jantasin et al., 2019c).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nThe authors would like to thank Dr. Sutin Chanabun for providing guidance and support for statistical analysis, further thanks for Asst. Prof. Niruwan Turnbull for the consultant of manuscript, the registered nurses who work at antenatal care clinics in 10 community hospitals in the Maha Sarakham province, and the pregnant adolescents who volunteered for this study. This study could not have been achievable without the dedicated support of all of them.\n\n\nReferences\n\nAbajobir AA, Maravilla JC, Alati R, et al.: A systematic review and meta-analysis of the association between unintended pregnancy and perinatal depression. J Affect Disord. 2016; 192: 56–63. PubMed Abstract | Publisher Full Text\n\nBolton HL, Hughes PM, Turton P, et al.: Incidence and demographic correlates of depressive symptoms during pregnancy in an inner London population. J Psychosom Obstet Gynaecol. 1998; 19(4): 202–209. PubMed Abstract | Publisher Full Text\n\nBunevicius A, Kusminskas L, Pop VJ, et al.: Screening for antenatal depression with the Edinburgh Depression Scale. J Psychosom Obstet Gynaecol. 2009; 30(4): 238–243. PubMed Abstract | Publisher Full Text\n\nCox JL, Holden JM, Sagovsky R: Detection of postnatal depression. Development of the 10-item Edinburgh Postnatal Depression Scale. Br J Psychiatry. 1987; 150(6): 782–786. PubMed Abstract | Publisher Full Text\n\nDa Costa D, Larouche J, Dritsa M, et al.: Psychosocial correlates of prepartum and postpartum depressed mood. J Affect Disord. 2000; 59(1): 31–40. PubMed Abstract | Publisher Full Text\n\nDennis CL, Ross LE, Grigoriadis S: Psychosocial and psychological interventions for treating antenatal depression. Cochrane Database Syst Rev. 2007; (3): CD006309. PubMed Abstract | Publisher Full Text\n\nEberhard-Gran M, Eskild A, Tambs K, et al.: Review of validation studies of the Edinburgh Postnatal Depression Scale. Acta Psychiatr Scand. 2001; 104(4): 243–249. PubMed Abstract | Publisher Full Text\n\nEvans J, Heron J, Francomb H, et al.: Cohort study of depressed mood during pregnancy and after childbirth. BMJ. 2001; 323(7307): 257–260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nField T: Prenatal Depression Risk Factors, Developmental Effects and Interventions: A Review. J Pregnancy Child Health. 2017; 4(1): pii: 301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrote NK, Bridge JA, Gavin AR, et al.: A meta-analysis of depression during pregnancy and the risk of preterm birth, low birth weight, and intrauterine growth restriction. Arch Gen Psychiatry. 2010; 67(10): 1012–1024. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHodgkinson SC, Colantuoni E, Roberts D, et al.: Depressive symptoms and birth outcomes among pregnant teenagers. J Pediatr Adolesc Gynecol. 2010; 23(1): 16–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHosmer DW Jr, Lemashow S: Applied Logistic Regression. 2nd ed. New York: A Wiley Interscience Publication. 2000. Reference Source\n\nHsieh FY, Bloch DA, Larsen MD: A simple method of sample size calculation for linear and logistic regression. Stat Med. 1998; 17(14): 1623–1634. PubMed Abstract | Publisher Full Text\n\nJantasin B, Yoosook W, Thaewpia S: Raw data - factors associated with depression. figshare. Dataset. 2019a. http://www.doi.org/10.6084/m9.figshare.10072898.v2\n\nJantasin B, Yoosook W, Thaewpia S: Demographic questionnaire and EPDS in English. figshare. Dataset. 2019b. http://www.doi.org/10.6084/m9.figshare.10073012.v1\n\nJantasin B, Yoosook W, Thaewpia S: Demographic questionnaire and EPDS in Thai. figshare. Dataset. 2019c. http://www.doi.org/10.6084/m9.figshare.10073009.v1\n\nKaewjanta N: Depression in teenage pregnancy: factors, affect and prevention. Journal of Nursing Science & Health. 2012; 35(1): 82–88. Reference Source\n\nKoniak-Griffin D, Walker DS, de Traversay J: Predictors of depression symptoms in pregnant adolescents. J Perinatol. 1996; 16(1): 69–76. PubMed Abstract\n\nLancaster CA, Gold KJ, Flynn HA, et al.: Risk factors for depressive symptoms during pregnancy: a systematic review. Am J Obstet Gynecol. 2010; 202(1): 5–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee DT, Chan SS, Sahota DS, et al.: A prevalence study of antenatal depression among Chinese women. J Affect Disord. 2004; 82(1): 93–99. PubMed Abstract | Publisher Full Text\n\nLimlomwongse N, Liabsuetrakul T: Cohort study of depressive moods in Thai women during late pregnancy and 6–8 weeks of postpartum using the Edinburgh Postnatal Depression Scale (EPDS). Arch Womens Ment Health. 2006; 9(3): 131–138. PubMed Abstract | Publisher Full Text\n\nLydsdottir LB, Howard LM, Olafsdottir H, et al.: The mental health characteristics of pregnant women with depressive symptoms identified by the Edinburgh Postnatal Depression Scale. J Clin Psychiatry. 2014; 75(4): 393–8. PubMed Abstract | Publisher Full Text\n\nMeltzer-Brody S, Bledsoe-Mansori SE, Johnson N, et al.: A prospective study of perinatal depression and trauma history in pregnant minority adolescents. Am J Obstet Gynecol. 2013; 208(3): 211.e1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Public Health: Statistics on Adolescent Births, Thailand 2015. Department of Health, Ministry of Public Health. 2017; 1: 11–146. Reference Source\n\nMuangpin S, Tiansawad S, Kantaruksa K, et al.: Northeastern Thai adolescents’ perceptions of being unmarried and pregnant. Pacific Rim Int J Nurs Res. 2010; 14(2): 149–161. Reference Source\n\nNasreen HE, Kabir ZN, Forsell Y, et al.: Prevalence and associated factors of depressive and anxiety symptoms during pregnancy: a population based study in rural Bangladesh. BMC Womens Health. 2011; 11: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNirattharadorn MK, Gennaro S, Vorapongsathorn T, et al.: Self-esteem, social support and depression in Thai adolescent mothers. Thai J Nurs Res. 2005; 9(1): 63–75. Reference Source\n\nOrr ST, Blazer DG, James SA, et al.: Depressive symptoms and indicators of maternal health status during pregnancy. J Womens Health (Larchmt). 2007; 16(4): 535–542. PubMed Abstract | Publisher Full Text\n\nOsok OJ: Depression and psychosocial risk factors associated with pregnant adolescent mixed method study based at Kangemi health centre. Nairobi University. 2016; [Cite 2018 september 15]. Reference Source\n\nPitanupong J, Liabsuetrakul T, Vittayanont A: Validation of the Thai Edinburgh Postnatal Depression Scale for screening postpartum depression. Psychiatry Res. 2007; 149(1–3): 253–259. PubMed Abstract | Publisher Full Text\n\nRoomruangwong C, Epperson CN: Perinatal depression in Asian women: prevalence, associated factors, and cultural aspects. Asian Biomedicine. 2011; 5(2): 179–193. Publisher Full Text\n\nSiegel RS, Brandon AR: Adolescents, pregnancy, and mental health. J Pediatr Adolesc Gynecol. 2014; 27(3): 138–150. PubMed Abstract | Publisher Full Text\n\nStapleton LR, Schetter CD, Westling E, et al.: Perceived partner support in pregnancy predicts lower maternal and infant distress. NIH public access. J Fam Psychol. 2012; 26(3): 453–463. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzilos GK, Zlotnick C, Raker C, et al.: Psychosocial factors associated with depression severity in pregnant adolescents. Arch Womens Ment Health. 2012; 15(5): 397–401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUNFPA: The state of Thailand’s population 2013: Motherhood in childhood facing the challenge of adolescent pregnancy . Thailand: Prang Priyatruk. 2014a. Reference Source\n\nUNFPA: The state of Thailand’s population Report 2013.2014b; [Cite 2018 April 19]. Reference Source\n\nUthaipaisanwong A, Rungruxsirivorn T, Roomruangwong C, et al.: Associated factors of prenatal depression among teenage pregnant women at King Chulalongkorn Memorial Hospital. J Med Assoc Thai. 2015; 98(5): 437–443. PubMed Abstract\n\nVacharaporn K, Pitanupong J, Samangsri N: Development of the Edinburgh Postnatal Depression Scale Thai version. J Mental Health Thai. 2003; 11(3): 164–169. Reference Source"
}
|
[
{
"id": "58099",
"date": "10 Feb 2020",
"name": "Felix Emeka Anyiam",
"expertise": [
"Reviewer Expertise Public Health",
"Population Health",
"Global Health",
"Epidemiological Research",
"Biostatistics",
"Open Research Data",
"Open Science",
"Data Science",
"Non-communicable diseases",
"Maternal and Child Health",
"Vulnerable Populations",
"Population at Risk",
"Reproductive Health",
"Data Management",
"Community-Based Research",
"HIV/AIDS Prevention",
"TB Prevention",
"Health Management and Health Promotion"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nThe article titled \"Predictive statistical model for the factors associated with prenatal depression among pregnant adolescents in Maha Sarakham province, Thailand\", is a cross-sectional study focused on adolescent pregnant mothers aged ≤19 years, and have an estimated gestational age of ≤18 weeks. The study is relevant, as it highlighted several risk factors associated with prenatal depression and some of which are: level of education, perceived support from husband and perceived relationship with husband. The study found a high prevalence of depression among this group of people which I think is a public health concern needing an urgent governmental intervention.\n\nReport:\nA. Title:\nThe title for this study is not appropriate as I did not see a predictive statistical modeling. Predictive modeling in statistics is more complex than what you have here. Sometimes, data mining and probability are applied in order to forecast outcomes. I think you should just leave the title as: Factors associated with prenatal depression among pregnant adolescents in Maha Sarakham province, Thailand\n\nB. Abstract:\nThe results section, the sentence with the words: “… and those with a perceived bad relationship with their husband or partner seven times higher.” should read: “…and those with a perceived bad relationship with their husband or partner had seven times higher .”\nC. Introduction:\n\nThe statement, “Adolescents can develop health problems during pregnancy that adversely affect both physical and psychological health (UNFPA, 2014b).” should read, “Adolescents can develop health problems during pregnancy that adversely affect both their physical and psychological health (UNFPA, 2014b).\n\nI do not think your introduction reference should begin with UNFPA, 2014b but instead UNFPA, 2014a. “a” should come before “b.” Please do switch the reference.\n\nI am not sure what the phrase “...adult women” means in the sentence: “Risks factors for depression among pregnant adolescents include not only those listed for adult women…” as you did not list any risk factors for adult women.\n\nThe statement, “ …and a history of emotion, sexual, and physical abuse..” should read, \" …and a history of emotional, sexual, and physical abuse.\n\n“Little research, however, has been conducted in northeastern Thailand, especially in Maha Sarakham province, where the rate of adolescent pregnancy is reported to be high.\" is a very strong statement and should be backed-up with literature.\n\nD. Results:\nYour Table 2 has 4 p-values but you only have 3 variables here you are associating. Please confirm and correct.\n\nIn Table 2, you also have 5 Adjusted OR. I think this should be just the 3 you mentioned in your text: 6.5, 5.1 and 7.4.\n\nE. Discussion:\n“As a matter of public health...” should read, “As a matter of public health concern”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5446",
"date": "29 Apr 2020",
"name": "Bhanwasa Jantasin",
"role": "Author Response",
"response": "Report:A. Title: The title for this study is not appropriate as I did not see a predictive statistical modeling. Predictive modeling in statistics is more complex than what you have here. Sometimes, data mining and probability are applied in order to forecast outcomes. I think you should just leave the title as:Factors associated with prenatal depression among pregnant adolescents in Maha Sarakham province, Thailand Answer:We discussed the title and prefer to preserve it as our statistical analysis (ROC = 73%) is plausible to forecast future outcomes.B. Abstract: The results section, the sentence with the words: “… and those with a perceived bad relationship with their husband or partner seven times higher.” should read: “…and those with a perceived bad relationship with their husband or partner had seven times higher.” Answer: we added the missing word according to the comment.C. Introduction: The statement, “Adolescents can develop health problems during pregnancy that adversely affect both physical and psychological health (UNFPA, 2014b).” should read, “Adolescents can develop health problems during pregnancy that adversely affect both their physical and psychological health (UNFPA, 2014b). Answer: we added the missing word according to the comment. I do not think your introduction reference should begin with UNFPA, 2014b but instead UNFPA, 2014a. “a” should come before “b.” Please do switch the reference. Answer: we revised the reference according to the comment. I am not sure what the phrase “...adult women” means in the sentence: “Risks factors for depression among pregnant adolescents include not only those listed for adult women…” as you did not list any risk factors for adult women Answer: We mean “pregnant adult women” and the risks factors are listed in the first sentence. We added the word ‘pregnant’ in front of ‘adult women’ to make it more clear. The statement, “ …and a history of emotion, sexual, and physical abuse..” should read, \" …and a history of emotional, sexual, and physical abuse. Answer: we added the missing word according to the comment. “Little research, however, has been conducted in northeastern Thailand, especially in Maha Sarakham province, where the rate of adolescent pregnancy is reported to be high.\" is a very strong statement and should be backed-up with literature. Answer: we added the reference according to the comment.D. Results: Your Table 2 has 4 p-values but you only have 3 variables here you are associating. Please confirm and correct. In Table 2, you also have 5 Adjusted OR. I think this should be just the 3 you mentioned in your text: 6.5, 5.1 and 7.4. Answer: we revised the table according to the comment.E. Discussion: “As a matter of public health...” should read, “As a matter of public health concern”. Answer: we added the missing word according to the comment."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1921
|
https://f1000research.com/articles/9-308/v1
|
29 Apr 20
|
{
"type": "Research Article",
"title": "Combinations of propolis and Ca(OH)2 in dental pulp capping treatment for the stimulation of reparative dentin formation in a rat model",
"authors": [
"Retno Pudji Rahayu",
"Nirawati Pribadi",
"Ira Widjiastuti",
"Nur Ariska Nugrahani",
"Nirawati Pribadi",
"Ira Widjiastuti",
"Nur Ariska Nugrahani"
],
"abstract": "Background: Caries in the dental pulp result in inflammation and damage to the pulp tissue. During inflammation of the pulp, various inflammatory mediators and growth factors are released, including IL-8, IL-10, TLR-2, VEGF and TGF-β through the NF-kB pathway. In the present study, therapy for pulpal caries was performed through pulp capping by giving a combination of propolis and calcium hydroxide (Ca(OH)2). This treatment was expected to stimulate the formation of reparative dentin as an anti-inflammatory material to prevent pulp tissue damage. Methods: 28 Wistar rats were divided into four groups and treated with Ca(OH)2 with or without the addition of propolis for either 7 or 14 days. Immunohistochemical examination was used to determine the expression of IL-8, IL-10, TLR-2, VEGF, TGF-β in the four treatment groups. Results: The group treated with a combination of propolis and Ca(OH)2 for 7 days showed that the expression of IL-10, IL-8, TLR-2, VEGF, TGF-β increased significantly compared to the treatment group treated with only Ca(OH)2. The expression of IL-10, TLR-2, TGF-β, VEGF increased in the treatment group treated with propolis and Ca(OH)2 for 14 days, while the expression of IL-8 in the decreased significantly. Conclusions: Administration of a combination of propolis and Ca(OH)2 has efficacy in the pulp capping treatment process because it has anti-bacterial and immunomodulatory properties. The results show that it is able to stimulate the process of pulp tissue repair through increased expression of IL-10, TGF-β, VEGF, TLR -2 and decreased expression of IL-8.",
"keywords": [
"Propolis",
"Ca (OH)2",
"IL-10",
"IL-8",
"TGF-ß",
"VEGF dan TLR-2"
],
"content": "Introduction\n\nCaries in the dental pulp result in inflammation and damage to the pulp tissue. During inflammation of the pulp, inflammatory mediators, including cytokines, regulate the activity of the immune response to local and systemic inflammation against the external stimuli1–3. NF-kB activation due to injury causes transcription and translational processes in cells, which results in pro-inflammatory cytokine production, such as IL-1β, IL-6 and TNF-α, initiating the immunological process that aids pulp tissue in overcoming inflammation3. NF-kB is considered as a prototypical inflammatory pathway and is used to target anti-inflammatory drugs, thus the expression of NF-kB can be investigated to measure the inhibition of proinflammatory cytokine production3,4.\n\nCalcium hydroxide (Ca(OH)2) is the gold standard for pulp capping treatment in tooth pulp inflammation but has a high pH (pH 12.5), which causes pulp tissue necrosis in direct contact with Ca(OH)25. Ca(OH)2 plays a role in the formation of reparative dentine, apexification, and intracanal medicament. It has anti-microbial properties (able to neutralize microbial products), inhibits root resorption and induces the formation of hard tissue6. The application of Ca(OH)2 with a certain concentration causes fibroblast cell death resulting in damage to the pulp tissue7. In addition, use of Ca(OH)2 in long term can cause physical changes; reparative dentine formation is not dense enough due to dentin bridge discontinuity leading to an area of necrosis called a \"tunnel defect\", whichcauses dentists to search for alternative materials, including natural herbal-based ingredients8. Propolis is a natural material produced by bees, consisting of balsam resin, beeswax, essential oils, and pollen from other organic materials. It may have a role in the process of pulp capping treatment and the healing process, due to its anti-bacterial, anti-fungal, anti-virus, anti-tumor, anti-oxidation, and immunomodulatory properties9. It has been reported that toxicity of propolis to fibroblast cells is low and is able to inhibit apoptosis and stimulate fibroblast cell proliferation10. In addition, propolis may be used as an alternative herbal ingredient for pulp capping treatment because it can reduce inflammation in pulp exposed to cariogenic material11. The inhibition of NF-kB by the active component of propolis causes a decrease in the expression of proinflammatory cytokine genes, such as IL-1, IL-6, IL-8, and TNF-α, leading to downregulation of TLR-2 in dental pulp12,13. The combination of Ca(OH)2 with propolis does not cause a toxic reaction, which was shown when evaluating the biocompatibility of Ca(OH)2 and propolis in the subcutaneous tissue of rats. It was found that both materials were able to reduce inflammation significantly through the role of IL-10, TGF-β, VEGF and was biocompatible with rat connective tissue11,14. Propolis contains caffeic acid phenethyl ester (CAPE), showing that propolis can function as an anti-inflammatory. However, to date research on pulp capping treatment for the stimulation of reparative dentin formation with a combination of Ca(OH)2 and propolis in an immunopathological study has not yet been performed. Therefore, this study aimed to find out if there was increased expression of IL-10, VEGF, TGF-β and TLR-2 as well as a decrease in pro-inflammatory cytokines IL-8 when using Ca(OH)2 in combination with propolis.\n\n\nMethods\n\nAll procedures performed in this study were ethical, and the study was approved by the Ethics Commission of the Dental and Oral Hospital of the Faculty of Dentistry, Airlangga University (002/HRECC.DHAU/XI/2019) with principal researcher as RPR. The study took place at the Department of Biochemistry, Medical Faculty, Airlangga University and Department of Biochemistry, Medical Faculty, Brawijaya University, which were certified to be ethical. All efforts were made to ameliorate any suffering of animals with the use of local anesthesia (lidocaine with Citoject syringe) to decrease pain in this treatment.\n\nAdult male Wistar rats aged 16–18 weeks, weight 150–250 grams, with healthy teeth and no caries, were kept in cages at room temperature (26°C) in light conditions, with food (Phokphan Hi-Provite) and water to drink available ad libithum. The rats were obtained from Biokimia Laboratories, Medical Faculty, Airlangga University. The number of rats obtained was calculated using the formula by Frederer: Sample = (r-1) (t-1), so that the total number of rats was 48. In each cage there were 7 rats and the rats were acclimatised to their cages for 1 week. The rats were kept in cages containing clean husk cushions and covered with a cover made of tenuous wire to function air circulation. Chaff was replaced three times a day to keep the cage clean.\n\nThe 48 Wistar strain rats were divided into eight groups (6 rats/group/time point): Control group, treated with aquadest (no drilling); negative control group, drilling only with no treatment; P1, treated with 0.625 μg Ca(OH)2 (Dycal from Dentsply); and P2, treated with a combination of propolis extract (natural raw bee) propolis extract powder (from Mellifera apis; concentration of 0.937 μg) with Ca(OH)2 (0.781 μg) in a ratio of 1:1.5.\n\nAnimals were randomly allocated to each group with random number generator. For each group, rats were given a number between 1 and 48. The first 6 numbers generated were in the positive control group, the next 6 in the negative control group and so forth.\n\nOn day 1 of the experiment, all groups of experimental animals were prepared for grade 1 cavity with a low speed round bur (0.84 mm diameter) to reach the right mandibular M1 pulp chamber. Rats were anesthetized using ketamin (ket A) dissolved in a sterile isotonic saline solution (0.2 ml/87 mg bb). The treatments were applied to the molar teeth of the rats as follows: group P1, Ca(OH)2 base was mixed with catalyst until it formed a paste-like consistency and this was applied to the molar teeth of the animal when it received a plastic filling; group P2, propolis extract powder was mixed with Ca(OH)2 powder and liquid until it formed a paste-like consistency.\n\nOn day 7, half the animals from each group (6/group) were anesthetised with ether and decapitated. Their jaws were removed and placed in a sample pot with 10% formalin buffer (pH 7.4). On day 14, the remaining animals (6/group) underwent the same procedures.\n\nThe dental tissue removed from the rats were kept in the sample pot with 10% formalin buffer (pH 7.4) for 24 hours. Subsequently, the tissue was decalcified using EDTA 10% at 37°C, which was replaced every day until the soft tissue broke down (this took up to 2 months). If the teeth were soft enough to be pricked with needle, then it is processed to make a paraffin block through the stages of dehydration, clearing, impregnation and embedding, as follows: dehydration, the dental tissue is kept in various percentages of alcohol (70, 80, 95, 96, 96, 96%) for 2 hours each percentage; clearing, dental tissue is placed in n-xylol for 4 hours; impregnation, the dental tissue is placed in solid paraffin for 4 hours; embedding, the dental tissue in the solid paraffin is placed into a mould base using tweezers. The mould base is filled with tissue on the cold plate and paraffin block ready to be cut. Using a microtome, tissue slices at a thickness of 0.5 cm were cut. These were stained with haematoxylin to look for macrophages, and were then prepared to check expressions of cytokines using the following kits: IL-10 (sc-52560 from Santa Cruz Biotechnology (Europe), VEGF (sc-7279 from Santa Cruz Biotechnology (Europe), IL-8 (BS3479 from Bioworld Technology), TGF-β (ab27969 from Abcam) and TLR-2 (pAb anti-TLR2 NB 200-536 from Novus Biologicals). Expression was looked at under a light binoculer microscope (Merk Olympus) at 400x magnification. For macrophages expressing the cytokines, cells that were brown were counted as positive.\n\nData analysis was completed with SPSS 16.0 version. Normality testing was done using Kolmogorov-Simrnov test, and a homogeneity test using Levene’s test. One-way ANOVA test used to assess the difference between groups, followed by post-hoc least significantt difference tests used for homogeneous data and Tamhane test used for non-homogeneous data. P value >0.05 was considered significant39,40.\n\n\nResults\n\nThe control group that was not treated with either Ca(OH)2 or propolis had higher VEGF expression than IL-10, TGF-β, IL-8, and TLR-2, while the negative control group (drilled but no treatment) had higher IL-8 expression than IL-10, TGF-β, VEGF, TLR-2. In the P1 group (Ca(OH)2 only) had higher VEGF expression than IL-8, TLR- 2, IL-10, TGF-β, while in group P2 (Ca(OH)2 and propolis combination) had higher IL-10 expression compared to VEGF, IL-8, TGF-β, and TLR-2 (Table 1).\n\nData were normally distributed, and homogeneity testing showed that IL-10 and VEGF were not homogeneous, while IL-8, TGF -β, TLR-2 were homogeneous. To see a significant difference using the One-way ANOVA and post-hoc testing showed that there were significant differences between the expression of IL-10, VEGF, IL-8, TGF-β and TLR-2 in the negative control group and the P2 group (combination of propolis extract with Ca(OH)2) (p = 0.00).\n\nThe control group (not treated) had IL-10 expression higher than VEGF, TGF-β, IL-8, and TLR-2, while the negative control group (drilled but not treated) had a higher expression of IL-8 than IL-10, TGF-β, VEGF, or TLR-2. In group P1 (Ca(OH)2 only) had higher IL-8 expression than VEGF, TLR-2, IL-10, TGF-β, while in P2 group (Ca(OH)2 and propolis combination) had higher VEGF expression compared to IL-10, IL-8, TGF-β, and TLR-2 (Table 2).\n\nData were normally distributed, and homogeneity testing showed that showed that VEGF and IL-8 expression were not homogeneous and IL-10, TGF-β and TLR-2 were homogenous. One-way ANOVA and post-hoc testing showed that there were significant differences between IL-10, VEGF, IL-8, TGF-β, and TLR-2 expression in the negative control group compared with the P2 group (combination of propolis extract with Ca(OH)2) (p = 0.00).\n\n\nDiscussion\n\nThis study used a combination of propolis and Ca(OH)2 as ingredients for the formation of reparative dentin. Propolis has advantages as an anti-inflammatory ingredient as it contains CAPE13,14. The anti-inflammatory properties possessed by propolis can reduce inflammation in the pulp chamber and can induce reparative dentin15. To find out whether propolis can be used as a bioproduct for stimulation of reparative dentin formation, this study tested the expression of the following cytokines: TLR-2, a membrane receptor that facilitates signaling pathway in the reparative dentin process16; IL-10, an anti-inflammatory cytokine17,18; VEGF, a protein that helps in the process of angiogenesis19,21; IL-8, a pro-inflammatory cytokine that can induce the formation of reparative dentin through the recruitment of neutrophils to repair damaged tissue damage22,23 ; and TGF-β, a regulator of cell proliferation, differentiation and reparative dentinogenesis24.\n\nThis study showed that on day 7, the group treated with a combination of propolis and Ca(OH)2 showed significantly increased expression of IL-10, VEGF, IL-8, TGF-β, and TLR-2 compared with the negative control. IL-10 expression was the highest among all the cytokines. Propolis has anti-inflammatory components, such as caffeic acid, which can inhibit the synthesis of eicosanoids from arachidonic acid and suppress the activity of COX-1 and COX-2 enzymes. Therefore, it inhibits the release of inflammatory mediators such as PGE-2 (prostaglandin), leukotrienes and thromboxanes, leading to the increasing expression of IL-10 expression with increasing dose of propolis23,24. The decrease in prostaglandin is caused by the inhibition of prostaglandin synthesis from arachidonic acid which is catalyzed by the transformation of the cyclooxygenase enzyme, which is produced by galangin compounds from flavonoids and CAPE. In addition, galangin from flavonoids are also able to inhibit leukotrienes from arachidonic acid through the enzyme lipoxygenase25,24.\n\nThe study showed that on day 14, there were changes in the expression of IL-10, TGF-β, and TLR-2 in the combination treatment group compared with the negative control group. The expression of IL-10, TGF-β, and TLR-2 in the treatment group treated with Ca(OH)2 alone without propolis was more significant compared with the negative control, whereas the expression of VEGF and IL-8 in the treatment group treated with a combination of propolis and Ca(OH)2 was significantly increased compared with the negative control. The combination treatment group showed the highest VEGF expression among other variables, while the Ca(OH)2 group showed that the expression of IL-10 was highest among the other variables. The 14th day is probably when the process of proliferation and angiogenesis has been initiated. In this research combination therapy of propolis and calcium hydroxide increases VEGF at 14 days. This could be due to the role of PDGF (platelet-derived growth factor), which is known as a promoter of proteoglycan and collagen formation. As local fibroblasts respond to PDGF by producing collagen and transforming it into myofibroblasts to increase wound contraction, fibroblasts also emit keratinocyte growth factor (KGF), which stimulate epithelialization from keratinocytes, and produce VEGF endothelial cell growth factors, and fibroblast growth factors (bFGF) to promote blood vessel growth27. IL-10 expression has also increased because Ca(OH)2 has high alkaline properties, which means that it will increase the pH for pulp healing if damaged as a high pH can neutralize acids in the inflammatory site28.\n\nCa(OH)2 is used as an intracanal medicament and is antibacterial. Ca(OH)2 is used because it is stable for a long time and can kill bacteria26,29. In previous studies, endodontic therapy using Ca(OH)2 showed that IL-1α as an indicator of pro-inflammatory cytokines decreased on day 7, indicating that there is an immune system response to reduce the inflammatory process so that the expression of IL-10 as an anti-cytokine -inflammation will increase26,30.\n\nCalcium hydroxide can induce odontoblast formation through increased VEGF, this is due to the occurrence of differentiation and viability of endothelial cells31,32. VEGF is also expressed by odontoblasts and sub-odontoblasts by large numbers of endothelial cells33. VEGF also has a key role in the physiological and pathological development of dentinogenesis and angiogenesis in healthy dental pulp33,34. In previous studies, odontoblast-like cells and undifferentiated pulp cells express high amounts of VEGF in vitro35. In the inflammatory process, day 7 shows an increase in VEGF after the formation of granulation tissue and decreases until day 1435,36. The VEGF mechanism is as an angiogenesis process through odontoblasts; LPS stimulated from bacteria present in the pulp can increase the permeability of the blood vessels so as to facilitate the process of neutrophil, lymphocyte and monocyte diapedesis and recruit new blood vessels around the carious tissue to increase immune defense37.\n\nThe reparative dentin stimulation process occurs after pulp damage which is then treated using pulp capping material in the form of calcium hydroxide Ca(OH)2 which is influenced by the nature of cytotoxicity and cytokine production ability34,35. Inflammatory cytokines such as IL-8 found in dental pulp will recruit neutrophils in case of infection from bacteria, which will cause acute inflammation. IL-8 also plays a role in determining the duration of the inflammatory process21.\n\nIn the combination treatment of propolis with Ca(OH)2 from the 7th day to the 14th day, the expression of IL-10, VEGF, TGF-β increased while IL-8 and TLR-2 decreased. Propolis activated the initial steps of the immune response by upregulating TLR-2 expression and the production of pro-inflammatory cytokines in the rats, modulating the mechanisms of innate immunity. TGF-β1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production37. These events are associated with activin receptor-like kinase-5, Smad2/3 and MEK/ERK signaling and TGF-β will inhibit TLR-2 mediated odontoblast pathway when inflammation occurs in the pulp chamber37,38. TGF-β is a signaling molecule that induces cell proliferation, chemotaxis and apoptosis in monocyte, epithelial, mesenchymal and neuron tissues. In the formation of reparative dentin, TGF-β has an important role in regulating cell proliferation, differentiation and reparative dentinogenesis23. The release of TGF-β from the dentinal matrix requires diffusion, which generally passes through the dentinal tubules to the pulp cell to activate the signaling pathway through TLR-2. Activation of TLR-2 will increase the effector of the innate immune system, including pro-inflammatory cytokines, chemokines, and anti-inflammatory cytokines to repair tissue damage39. This is consistent with the results of this research showing that with the combination of propolis with Ca(OH)2 TGF-β expression is higher than in the treatment group with only Ca(OH)2. TGF-β is secreted by odontoblasts, and its expression is increased in carious lesions. TGF-β is proinflammatory during the initial stages of inflammation, and anti-inflammatory in later stages. Proinflammatory effects of TGF- β include immune cell recruitment and induction of matrix metalloproteinase secretion, and stimulation of the accumulation of immature dendritic cells in odontoblast and subodontoblast layers of the pulp horn close to the lesion in strategic locations to encounter foreign antigens entering the dentinal tissue14,39.\n\nIL-8 is expressed continuously by odontoblast cells. IL-8 plays a role in neutrophil recruitment in cases of inflammation in the pulp to support the healing process39. In this study, IL-8 was expressed at a lower in the propolis and Ca(OH)2 combination treatment group compared with the group treated with Ca(OH)2 only. This illustrates that the administration of a combination of propolis and Ca(OH)2 can repair pulp tissue damage through the formation of reparative dentine. On the 14th day with propolis and Ca(OH)2 combination, VEGF expression was high so that it can help the process of angiogenesis in the dental pulp to be faster, while the group with only Ca(OH)2 has anti-inflammatory properties and high IL-10 expression so as to reduce inflammation which occurs in the pulp chamber.\n\n\nConclusion\n\nCombination therapy of propolis with Ca(OH)2 could be considered as a product for pulp capping treatment that can stimulate the formation of reparative dentin.\n\n\nData availability\n\nHarvard Dataverse: Combinations of propolis and Ca(OH)2 in dental pulp capping treatment for the stimulation of reparative dentin formation in a rat model, https://doi.org/10.7910/DVN/SLV60A41\n\nThis project contains the following underlying data:\n\n- Values for immunohistochemistry for all groups for 7-day treatment\n\n- Values for immunohistochemistry for all groups for 14-day treatment\n\n- Figure S1. Mean of IL-10, VEGF, IL-8, TGF-β and TLR-2 expressions on the 7th day.\n\n- Figure S2. Mean of IL-10, VEGF, IL-8, TGF-β and TLR-2 expressions on the 14th day.\n\n- Figure S3. Immunohistochemical examination results from VEGF. A. Control, B. Negative Control, C. Therapy with Ca(OH)2, D. Therapy with a combination of propolis and Ca(OH)2.\n\n- Figure S4. Results of immunohistochemical examination of IL-10. A. Control, B. Negative Control, C. Therapy with Ca(OH)2, D. Therapy with a combination of propolis and Ca(OH)2.\n\n- Figure S5. Immunohistochemical examination results from TGF-β. A. Control, B. Negative Control, C. Therapy with Ca(OH)2, D. Therapy with a combination of propolis and Ca(OH)2.\n\n- Figure S6. Results of immunohistochemical examination of TLR-2. A. Control, B. Negative Control, C. Therapy with Ca(OH)2, D. Therapy with a combination of propolis and Ca(OH)2.\n\n- Figure S7. Results of immunohistochemical examination of IL-8. A. Control, B. Negative Control, C. Therapy with Ca(OH)2, D. Therapy with a combination of propolis and Ca(OH)2.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "References\n\nParolia A, Kundabala M, Rao NN, et al.: A comparative histological analysis of human pulp following direct pulp capping with Propolis, mineral trioxide aggregate and Dycal. Aust Dent J. 2010; 55(1): 59–64. PubMed Abstract | Publisher Full Text\n\nWidjiastuti I, Suradita K, Saraswati W: The Expressions of NF-Κb and TGFβ-1 on Odontoblast-like cells of Human Dental Pulp Injected with Propolis Extracts. Dental Journal: Majalah Kedokteran Gigi Airlangga University. 2014; 47(1): 13–18. Publisher Full Text\n\nLawrence T: The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009; 1(6): a001651. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJanebodin K, Horst OV, Osathanon T: Dental pulp respons to pulp capping materials and biosktif molecules. Cu Dent J. 2010; 33: 229–248. Reference Source\n\nDianat O, Azadnia S, Mozayeni MA: Toxicity of calcium hydroxide nanoparticles on murine fibroblast cell line. 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PubMed Abstract | Publisher Full Text\n\nRohman MS, Endah K, Dwi S, et al.: Inhibition of Nf-kB activation by CAPE (Caffeic Acid Phenethyl Ester) an Active Component of Honeybee Hives, in Oxldl-Treated Huvec’s (Human Umbilical Vein Endothelial Cells). Jurnal Kedokteran Brawijaya. 2006; 12(1): 1–13.\n\nSahamanta CG, Arzate SG, Flores C, et al.: Antimicrobial activity with mixture of calcium hydroxide and propolis. Int J Pharma Bio Sci. 2011; 2(4): 203–210. Reference Source\n\nMori GG, Rodrigues Sda S, Shibayama ST, et al.: Biocompatibility of a calcium hydroxide-propolis experimental paste in rat subcutaneous tissue. Braz Dent J. 2014; 25(2): 104–8. PubMed Abstract | Publisher Full Text\n\nS VK: Propolis in dentistry and oral cancer management. N Am J Med Sci. 2014; 6(6): 250–259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWięckiewicz W, Miernik M, Więckiewicz M, et al.: Review article: Does propolis help to maintain oral health? 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PubMed Abstract | Publisher Full Text\n\nTavares F, de Brito LC, Henriques LC, et al.: Effects of calcium hydroxide on cytokine expression in endodontic infections. J Endod. 2012; 38(10): 1368–1371. PubMed Abstract | Publisher Full Text\n\nKhan AA, Sun X, Hargreaves KM: Effect of calcium hydroxide on proinflammatory cytokines and neuropeptides. J Endod. 2008; 34(11): 1360–1363. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVirtej A, Løes S, Iden O, et al.: Vascular endothelial growth factors signalling in normal human dental pulp: a study of gene and protein expression. Eur J Oral Sci. 2013; 121(2): 92–100. PubMed Abstract | Publisher Full Text\n\nBotero TM, Son JS, Vodopyanov D, et al.: MAPK signaling is required for LPS-induced VEGF in pulp stem cells. J Dent Res. 2010; 89(3): 264–269. PubMed Abstract | Publisher Full Text\n\nShi S, Gronthos S: Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res. 2003; 18(4): 696–704. PubMed Abstract | Publisher Full Text\n\nNakanishi Y, Izumi K, Yoshizawa M, et al.: The expression and production of vascular endothelial growth factor in oral mucosa equivalents. Int J Oral Maxillofac Surg. 2007; 36(10): 928–933. PubMed Abstract | Publisher Full Text\n\nZarei M, Jafarian AH, Harandi A, et al.: Evaluation of the expression of VIII factor and VEGF in the regeneration of non-vital teeth in dogs using propolis. Iran J Basic Med Sci. 2017; 20(2): 172–177. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrsatti CL, Missima F, Pagliarone AC, et al.: Propolis immunomodulatory action in vivo on Toll-like receptors 2 and 4 expression and on pro-inflammatory cytokines production in mice. Phytother Res. 2010; 24(8): 1141–6. PubMed Abstract | Publisher Full Text\n\nLin PS, Chang HH, Yeh CY, et al.: Transforming growth factor beta 1 increases collagen content and stimulate procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling. J Formos Med Assoc. 2017; 116(5): 351–358. PubMed Abstract | Publisher Full Text\n\nHorst OV, Tompkins KA, Coats SR, et al.: TGF-beta1 Inhibits TLR-mediated odontoblast responses to oral bacteria. J Dent Res. 2009; 88(4): 333–337. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarges JC, Alliot-licht B, Renard E, et al.: Dental Pulp Defence and Repair Mechanism in Dental Caries. Mediators Inflam. 2015; 2015: 230251. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDodge Y: The Concise Encyclopedia of Statistics: Least Significant Difference Test. Springer: New York. 12. Publisher Full Text\n\nRahayu RP: \"Combinations of propolis and Ca(OH)2 in dental pulp capping treatment for the stimulation of reparative dentin formation in a rat model\".2020. http://www.doi.org/10.7910/DVN/SLV60A"
}
|
[
{
"id": "62925",
"date": "07 May 2020",
"name": "Guang Hong",
"expertise": [
"Reviewer Expertise Biomaterials and tissue 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\nThis paper needs minor revision. I would make the following comments.\n\nIntroduction:\nPlease add the hypothesis of your study in this section.\nMethods:\nIn your study, you use the 16-18 weeks rat. I think it is a little bit old, how do you think? Please add the reason why you choose this age rat in the discussion section.\nDiscussion:\n\nThe answer to the hypothesis of this study should be included in the discussion section.\n\nNatural ingredients are difficult to obtain stable properties. Please add the discussion regarding this issue in the discussion section.\n\nPlease add the clinical implications of your study and the clinical significance of the findings in the discussion section.\n\nWhat is the limitation of this study? You should indicate in this section.\n\nFigure:\nFig. 1 and Fig. 2: Please add the result of the statistical analysis.\n\nFig. 3 to Fig. 7: Please use arrows to indicate the point that you want to explain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "65377",
"date": "06 Jul 2020",
"name": "Mohd Farhan Hanif Reduan",
"expertise": [
"Reviewer Expertise My research on the wound healing and toxicological study using an animal model in particular to pathological evaluation."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewer comment:\nCombinations of propolis and Ca(OH)2 in dental pulp capping treatment for the stimulation of reparative dentin formation in a rat model.\nReviewer comments: In general, the research is well conducted and the findings are worth considering for publication. The paper needs a minor revision as below:\nConclusion of the abstract, please omit the “antibacterial property” as the research conducted not based on the antibacterial study.\n\nParagraph 1: “NF-kB activation due to injury causes transcription and translational processes in cells, which results in pro-inflammatory cytokine production, such as IL-1β, IL-6 and TNF-α, initiating the immunological process that aids pulp tissue in overcoming inflammation” - the reviewer suggest confirming this statement as pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α are promoting inflammation, not overcome the inflammation, so need clarification on this statement.\n\nMethodology – in the animal section, the authors need to replace the “biokimia” term into the English term.\n\nMethodology – in the treatment section, the authors need to highlight the experiment is separated into two periods of time; 7 and 14 days at the initial, easier for the reader to capture, the authors need to explain why two different concentrations of calcium hydroxide were selected in the discussion.\n\nMethodology – in the immunohistochemistry section, please use a past tense to describe the terms, few in the present tense, the authors need to explain in detail on the evaluation of the expression of cytokines like the intensity of the brownish expression and etc. If possible, include references on the method of immunohistochemistry. It is because immunohistochemistry is a major part of this study.\n\nResults – The authors are suggested to include the statistical indication in Tables 1 and 2. It will help the reader to understand the table faster rather than read in the sentences. Please put arrows in the figures.\n\nDiscussion – Paragraph 2; “In addition, galangin from flavonoids are also able to inhibit leukotrienes from arachidonic acid through the enzyme lipoxygenase”- authors need to state what is the source of galangin, is it from propolis?\n\nDiscussion – Paragraph 3; “The 14th day is probably when the process of proliferation and angiogenesis has been initiated”, need to improvise the statement as the proliferation and angiogenesis are at the peak in the 14th day.\n\nDiscussion – Paragraph 5: “Ca(OH)2 is used as an intracanal medicament and is antibacterial. Ca(OH)2 is used because it is stable for a long time and can kill bacteria”. The authors are suggested to further discuss the details on the effect of calcium hydroxide as caries always related to bacterial infection.\n\nDiscussion – Paragraph 8: The authors need to add elaboration on the anti-inflammatory activity of TGF at the late stage.\n\nConclusion – the mechanism on the stimulation of the formation of reparative dentin needs to be addressed based on the present 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": []
},
{
"id": "63240",
"date": "17 Jul 2020",
"name": "Noengki Prameswari",
"expertise": [
"Reviewer Expertise Bone remodeling",
"herbal medicine",
"physiology",
"orthodontic"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe sample size mentioned in abstract methods and full-text methods are very vague. They should be rewritten for better clarity.\n\nMethods: you mention that this treatment for stimulating the formation of reparative dentine. What is the clinical parameter for showing that your treatment (dental pulpcapping) has an effect on the formation of reparative dentine?\n\nDiscussion: The explanation about the role of natural ingredient in Propolis and its combination with Ca(OH)2 as dental pulpcapping to stimulate the formation of reparative dentine is not clear.\n\nConclusion: you should state the difference result of pulpcapping treatment in 7 days and 14 days, and also the mechanism.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-308
|
https://f1000research.com/articles/9-257/v1
|
14 Apr 20
|
{
"type": "Opinion Article",
"title": "Gilles de la Tourette Syndrome: advice in the times of COVID-19",
"authors": [
"Mary M. Robertson",
"Valsamma Eapen",
"Renata Rizzo",
"Jeremy S. Stern",
"Andreas Hartmann",
"Mary M. Robertson",
"Valsamma Eapen",
"Renata Rizzo",
"Jeremy S. Stern"
],
"abstract": "The novel coronavirus disease (COVID-19) was identified as the cause of an outbreak of respiratory disease in China at the end of 2019. It then spread with enormous rapidity and by mid-March 2020 was declared a world pandemic. Gilles de la Tourette Syndrome (GTS) is a childhood-onset neurodevelopmental disorder with a worldwide prevalence of about 1% of the population. The clinical symptoms include multiple motor and one or more phonic (vocal) tics. Germane to this communication is that 85% of patients with GTS have associated psychiatric co-morbidities, many of which are being exacerbated in the current global health crisis. In addition, several symptoms of GTS may mimic COVID-19, such as a dry cough and sniffing (phonic tics), while other symptoms such as spitting, inappropriate touching of others and “non-obscene socially inappropriate symptoms” can potentially get patients with GTS into trouble with the law. We suggest that a clear explanation of the COVID-19 illness and GTS is important to enable colleagues of various specialities who tend to patients with GTS. It is important to acknowledge at the outset that the information available on the COVID-19 pandemic changes daily, including cases infected, deaths reported, and how various national health systems are planning and or coping or not. It is fair to say that having read the current medical and lay press we conclude that it is not easy to reassure our patients with absolute certainty. However, notwithstanding that, we hope our documentation is of some assistance.",
"keywords": [
"Gilles de la Tourette syndrome",
"COVID-19",
"tics",
"anxiety",
"OCD",
"ADHD",
"confinement"
],
"content": "Aetiology: COVID-19 (coronavirus)\n\nCoronaviruses are a group of viruses that mostly result in mild illnesses, similar to the “common cold”, targeting the upper respiratory tract. However, certain types of coronaviruses can affect the lower airway, causing serious illnesses such as bronchitis or pneumonia. Coronaviruses have very large single stranded RNA genomes (approximately 26-32 KB). Coronavirus particles are surrounded by a fatty outer layer called an “envelope” and usually are spherical when visualised under an electron microscope, with a “corona” of club-shaped spikes on their surface (Wu et al., 2020). Most people become infected with coronaviruses at some point in their lives, but the majority of these are harmless. The novel coronavirus, which causes the COVID-19 illness, is different, and can be extremely serious.\n\nThe virus that causes COVID-19 is known as SARS-Cov-2 (Severe Acute Respiratory Syndrome Corona Virus 2). It appears to have emerged in Wuhan, Hubei Province, China. The viruses first inhabited animals such as bats and then “jumped” to humans: this occurred in China in late 2019, from whence it has spread worldwide (Zhu et al., 2020). Coronaviruses replicate their RNA genomes using enzymes called RNA-dependent RNA polymerases, which are prone to errors, but genomic analysis to date suggests that SARS-Cov-2 is mutating slowly, thus reducing the chance of it changing to become deadlier (Wu et al., 2020). It is worth mentioning that since 2003, viral diseases have caused major epidemics. These include the coronaviruses, which have caused multiple major public health events that resulted in global epidemics such as severe acute respiratory syndrome (SARS; or “bat SARS”), Middle East respiratory syndrome (MERS) and the current coronavirus disease (COVID-19) (Kandeel et al., 2020). In addition, with a growing threat to maternal-foetal health, the Zika viral illness began in 2009 with devastating consequences and continues to spread (Towers et al., 2018). Finally, in some parts of the globe, cases of swine flu (H1N1 virus) are still on the rise such as in India, where in 2020 alone around 1500 cases have been reported to date, with 28 deaths in the period from 1 January to 1 March 2020.\n\n\nClinical features: COVID-19\n\nCOVID-19 can affect individuals of any ages and clinical symptoms may vary. The main recognised clinical features of COVID-19 include fever (52% of younger patient, 89% of older patients), cough (44% younger, 77% older; usually dry, but can be expectorant), fatigue or tiredness; more severe cases get pneumonia. In mild cases people may have only a runny nose or sore throat. Patients may also have gastro-intestinal symptoms such as nausea, diarrhoea and anorexia. It does appear that older people, on the whole, are more severely affected. Individuals with pre-existing co-morbid conditions also appear to be particularly at risk: these illnesses include hypertension, diabetes, cardiovascular disease, and malignancy (Mao et al., 2020; Wan et al., 2020; Zhang et al., 2020a; Zhang et al., 2020b; Zhang et al., 2020c; Zheng et al., 2020a). There is also recent evidence that anosmia or loss of smell is a symptom of COVID-19 and can even be the presenting symptom (Hopkins & Kumar, 2020; Lüers et al., 2020). Recent unpublished results in a series of 417 patients suggest rates of >80%, and a large European multicentre trial is currently being undertaken.\n\nSeverity has been described in various ways. Patients with abnormal chest images are older, with a higher rate of pre-existing conditions, lower rate of exposure history, and a longer time between onset and confirmation than patients without pneumonia. In addition, patients with more severe symptoms have a higher rate of fever, expectorant cough and headache, lower lymphocytes, albumin and serum sodium, and a higher total bilirubin, c-reactive protein, creatine kinase and lactate dehydrogenase, (Zhang et al., 2020c). Muscle aches, headache, shortness of breath and nausea/vomiting are also associated with disease severity. Also, biomarkers such as lower lymphocyte levels in addition to raised leukocytes, D-dimer levels, procalcitonin and serum creatinine, as well as higher radiograph score at admission are predictive factors for a severe/critical subtype (Zhang et al., 2020c). Finally, it has been shown that elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict progression in COVID-19 patients (Zheng et al. 2020b). In the most severe cases, patients go into respiratory and multi-organ failure. It must be borne in mind, however, that mortality has also been reported in children, adolescents and young adults. Thus, it does appear that no one is immune or not at risk.\n\n\nAetiology and epidemiology: Gilles de la Tourette Syndrome\n\nGilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder that has aetiological contributions from genetic influences in pregnancy and birth difficulties, and immunological variables (Robertson et al., 2017). Whereas the PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections) hypothesis is increasingly being discarded, an immunological trigger is possible in a subset of patients with GTS as they might show differences in immune responses compared to controls (Martino et al., 2015); however, how these might possibly affect response to viral infections and, more specifically SARS-Cov-2, remains as yet unknown (see below).\n\n\nClinical features: Gilles de la Tourette syndrome\n\nThe generally recognised diagnostic criteria according to the DSM-5 for GTS are multiple motor and at least one or more vocal (phonic) tics over the course of at least 12 months in a patient less than 18 years old (APA, 2013). The phenotypic spectrum also includes chronic (persistent) motor or vocal tic disorder and transient (provisional) tic disorder. The most common motor tics are facial (such as eye blinking and grimacing), whereas the most common phonic tics are coughing, sniffing and throat clearing (Robertson et al., 2017). Individuals may have the relatively rare (around 10%) but “press-worthy” symptom of coprolalia (involuntary swearing).\n\nNeuropsychiatric comorbidities are common (around 85% of patients) and include obsessive-compulsive disorder or behaviours (OCD/OCB), attention-deficit hyperactivity disorder (ADHD), learning disabilities, depression, anxiety, and autism (Rizzo et al., 2014; Robertson et al., 2017).\n\nIt is also worth noting that quality of life in patients with GTS has been demonstrated to be reduced (Elstner et al., 2001; Eddy et al., 2011). In addition, Eapen et al. (2016) reviewed the literature, pointing out that both tics and comorbidities in GTS may result in poorer psychosocial functioning. Much of the stigma and social maladjustment associated with GTS resulting in exclusion, bullying and discrimination, can be largely caused by misperceptions of the disorder by teachers, peers and the wider community. Thus, in the current “COVID-19 climate,” we must be mindful to remain attuned to the needs of our patients with GTS.\n\n\nGilles de la Tourette syndrome in the context of COVID-19\n\nPotential consequences of the COVID-19 pandemic on patients with GTS can be classified according to four axes:\n\n1. Anxiety related to the pandemic\n\n2. Confinement/quarantine\n\n3. Alterations in tics and behaviours specific to GTS\n\n4. Neurotropic effects of SARS-Cov-2\n\nAnxiety is a frequent comorbidity in GTS, manifesting both as generalized anxiety as well as specific phobias (Martino et al., 2017). Obviously, the relentless and generally starkly pessimistic media coverage on COVID-19 is likely to exacerbate these anxieties. In young children, their comprehension of the situation is limited, but the transmission of parental anxiety is a possible concern, either because of the epidemic itself and/or the economic consequences they face.\n\nOCD is an equally frequent comorbidity in GTS. In the current era of governmental and public health instructions designed to slow the spread of COVID-19, patients with contamination OCD are at particular risk of experiencing conflicting demands since their baseline cognitive-behavioural (CBT) training (if applicable) asks for less hand washing, the exact opposite of what is now being recommended: this may result in cognitive dissonance which may turn close to unbearable. Obsessions relating to death (one’s own or loved ones) will likely also rise, as well as morbid ruminations (Robertson & Cavanna , 2007), and possibly phenomena typical of GTS such as symmetry and ‘just right’ behaviours.\n\nIn addition, anxiety may trigger an increase in tic severity. It must be explained to patients and parents that this does not reflect an aggravation of GTS per se but is contextual, and thus likely reversible in the foreseeable future. An exacerbation of baseline tics in general may not be inevitable. In normal practice patients sometimes even report a reduction of GTS symptoms in association with major anxiety-provoking life events, although the reverse is conventionally more often true. Aside from OCD, entire populations are now undergoing some kind of “life event”, no doubt to be the subject of major study in due course.\n\nThe psychological sequelae of confinement and quarantine following the COVID-19 outbreak have been well reviewed recently (Brooks et al., 2020). The authors reviewed the psychological impact of quarantine using three data bases and 3166 papers out of which 24 were included. Most studies reviewed reported negative psychological effects including post-traumatic stress symptoms, confusion and anger. Stressors included longer quarantine duration, infection fears, frustration, boredom, inadequate supplies, inadequate information, financial loss and stigma. Some researchers suggested a long-lasting impact.\n\nAs with anxiety – since confinement and quarantine are anxiety-triggering in their own right – an increase in tic severity can be expected. Moreover, behavioural abnormalities will likely also be exacerbated. First comes ADHD, present in 50–70% of patients with GTS (Hirschtritt et al., 2015). In particular, the hyperactive phenotype makes confinement and quarantine hard to bear as physical energy cannot be released. It is also likely that rage attacks/explosive outbursts, as well as oppositional behaviour – which largely depend on the presence of comorbid ADHD (Müller-Vahl et al., 2020) – will put an increasing strain on families. Reducing screen time – a frequent trigger of explosive outbursts – follows a paradoxical injunction similar to hand washing since it is more difficult to enforce in conditions of confinement and quarantine, added to which comes reduced schooling time. Yet, this is a potential conflict for parents as allowing children and adolescents to use screens more abundantly than usual may be the only way to keep the peace for the time being. Nevertheless, even in instances where increased screen time cannot be avoided, it is still possible to ensure that the content of what is being watched is age appropriate, engaging/interactive and meaningful. With regard to ADHD and impulsivity, following the rules (hygiene and social distancing) may be particularly difficult. Note that behavioural abnormalities related to ADHD not only concern children but also adults, especially maladaptive behaviours (Haddad et al., 2009). Home schooling may further penalize children with learning disabilities, frequent in GTS. Lastly, a hallmark of GTS is individual variation. We have also heard from adult patients whose tics are usually worse when they are out in society and less intrusive at home; some have described being in self-isolation with families or alone and finding their tics relatively settled.\n\nAs outlined in the two previous sections, many problems that patients with GTS face in the context of COVID-19 are related to their comorbidities, with an increase in tics the only outcome specific to their underlying disorder. However, certain nosographic features unique to GTS must also be considered.\n\nFirstly, non-obscene socially inappropriate symptoms (NOSIS) (Eddy & Cavanna, 2013; Kurlan et al., 1996) which lead patients to display behaviours at odds with what is socially acceptable and can also be referred to as provocative and transgressive. Patients told us of developing new coughing tics, with a particular urge in public. Other examples to be expected in times of COVID-19 may manifest as spitting, sneezing into one’s hands, not maintaining social distancing, or attempting to shake hands (personal observations). Touching other people is a not an uncommon complex motor tic but in the “COVID-19 climate” could also represent NOSIS (see Table 1). Note that these behaviours can also fall into the risk-seeking/impulsive phenotype often present in patients with GTS (Wright et al., 2012). Furthermore, echolalia and echopraxia may lead patients with GTS to copy other people’s coughing. In case of palilalia and palipraxia (repeating their last actions), a first ‘real’ cough may be repeated over and over again without any underlying respiratory need.\n\nTo date, little is known about the potential neurological effects of SARS-Cov-2 but these have been suggested (Baig et al., 2020) and there is an evolving observation of high rates of anosmia and hypogeusia even in mild infection which may indicate neurotropism (Hopkins & Kumar, 2020; Lüers et al., 2020). Undoubtedly, this literature will grow over the upcoming months and years. As immune function may be altered (even though this does not necessarily mean diminished or compromised) in GTS, one may wonder whether our patients bear a special risk of sequelae. Intuition however dictates that this is highly unlikely as viral CNS infections have a higher probability to target people with neurodegenerative disease, or even causing it, as was the case after the Spanish influenza pandemic and resulting Encephalitis Lethargica (Hoffman & Vilensky, 2017). In neurodevelopmental disorders such as GTS – and this also holds true for its comorbidities which are based on circuit dysfunctions – it is, at present, hard to see how a viral infection, and more specifically SARS-Cov-2, could lead to lasting CNS sequelae specific to GTS (for an evolving online resource, see here). However, only time will tell.\n\n\nManagement and treatment\n\nAccurate recognition of the symptoms of coughing or sniffing as related to GTS as opposed to COVID-19 is critical in providing the right intervention and support. Similarly, spitting or forced touching etc. can be part of tic-related behaviours that may get the patient in trouble with the law. Hence awareness and appropriate support (e.g. the GTS patient may have to carry a card, informing authorities and other appropriate people about the condition) is important. Further, accurate identification and treatment of tics and comorbidities should continue as usual and according to established guidelines, if possible (Pringsheim et al., 2019). It is likely that medications will need to be increased probably transiently until the COVID-19 crisis resolves. That is obviously true for anti-tic medication as it is for psychostimulants (for ADHD) and SSRIs (selective serotonin reuptake inhibitors; for anxiety and OCD). There are currently unprecedented challenges in healthcare delivery and some of the specialists who would normally give this advice in many countries may be unavailable for a time due to redeployment, potentially causing further stress to families. Also, CBT for tics (van de Griendt et al., 2013) should be maintained by videoconferencing whenever possible (Andrén et al., 2019). The same applies to supportive psychotherapy for those who suffer from anxiety, depression, and loneliness.\n\nThe National Patient Advocacy Groups for GTS in the UK and USA have published factual information and advice for individuals with GTS, their families and health-care professionals. These represent invaluable resources in their own right and, moreover, underline a sense of (global) community that is vital in these times.\n\nConcerning the points raised above, we propose the following measures:\n\nReduce media coverage to curb anxiety.\n\nLimit parental anxiety (or the expression thereof) as much as possible.\n\nIn patients with contamination OCD, relax CBT rules in collaboration with the therapist. Insist that this is a temporary measure.\n\nExplain, if necessary, that tic increase does not reflect an aggravation of the primary disorder but is contextual, and thus transient.\n\nReduce screen time reasonably (i.e. without triggering rage attacks and to avoid excessive boredom), but more importantly focus on the content of what is being watched.\n\nUse outdoor activities as much as possible and legally acceptable, especially when ADHD is present, or engage in activities that can be done indoors.\n\nInsist on upholding current rules (hygiene, social distancing) firmly but also gently.\n\nIf patients get in conflict with the law or just other individuals because of NOSIS, echo- or palipraxia (i.e. abundant coughing in public or displaying at-risk behaviours), attempts should be made to resolve the issue by explaining these symptoms. In some cases, “urgency cards” can also be of great help (see here and here).\n\nFor health-care professionals: keep up outpatient clinics by telephone or videoconferencing as much as possible. The latter may prove useful even after the epidemic will have abated.\n\nFinally, there are no indications that SARS-Cov-2 infections might lead to central nervous system sequelae for patients with GTS in the intermediate or long term. Neither does GTS alone put individuals in a higher risk group for general consequences of infection. If necessary, reassurance is needed in this matter.\n\n\nConclusion\n\nGTS, as a complex neuropsychiatric disorder, offers many angles of attack for the current COVID-19 pandemic and its consequences (social distancing, home schooling, confinement/quarantine, and living in a general climate of fear). Also, some patients might expose themselves to social conflict because of behavioural features specific to GTS. Thus, they need our reassurance and help in these difficult times, supported by a network of informed health-care professionals, although already rare in ‘normal’ times. Ideally, specialist medical services will be able to organise in the face of acutely changing roles in hospitals to provide advice when needed, to avoid a feeling of professional abandonment (which obviously is not special to patients with GTS). With this short paper, we hope to have contributed a few small pieces to the puzzle which will hopefully help us navigate through the upcoming weeks and months.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nAPA: Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Washington: American Psychiatric Association; 2013. Reference Source\n\nAndrén P, Aspvall K, Fernández de la Cruz L, et al.: Therapist-guided and parent-guided internet-delivered behaviour therapy for paediatric Tourette's disorder: a pilot randomised controlled trial with long-term follow-up. BMJ Open. 2019; 9(2): e024685. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaig AM, Khaleeq A, Ali U, et al.: Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host-Virus Interaction, and Proposed Neurotropic Mechanisms. ACS Chem Neurosci. 2020; 11(7): 995–998. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–920. PubMed Abstract | Publisher Full Text\n\nEapen V, Cavanna AE, Robertson MM: Comorbidities, Social Impact, and Quality of Life in Tourette Syndrome. Front Psychiatry. 2016; 7: 97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEddy CM, Rizzo R, Gulisano M, et al.: Quality of life in young people with Tourette syndrome: a controlled study. J Neurol. 2011; 258(2): 291–301. PubMed Abstract | Publisher Full Text\n\nEddy CM, Cavanna AE: On being your own worst enemy: an investigation of socially inappropriate symptoms in Tourette syndrome. J Psychiatr Res. 2013; 47(9): 1259–63. PubMed Abstract | Publisher Full Text\n\nElstner K, Selai CE, Trimble MR, et al.: Quality of Life (QOL) of patients with Gilles de la Tourette's syndrome. Acta Psychiatr Scand. 2001; 103(1): 52–9. PubMed Abstract | Publisher Full Text\n\nHaddad AD, Umoh G, Bhatia V, et al.: Adults with Tourette's syndrome with and without attention deficit hyperactivity disorder. Acta Psychiatr Scand. 2009; 120(4): 299–307. PubMed Abstract | Publisher Full Text\n\nHopkins C, Kumar N: Loss of sense of smell as marker of COVID-19 infection. Reference Source\n\nHirschtritt ME, Lee PC, Pauls DL, et al.: Lifetime prevalence, age of risk, and genetic relationships of comorbid psychiatric disorders in Tourette syndrome. JAMA Psychiatry. 2015; 72(4): 325–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffman LA, Vilensky JA: Encephalitis lethargica: 100 years after the epidemic. Brain. 2017; 140(8): 2246–2251. PubMed Abstract | Publisher Full Text\n\nLüers JC, Klußmann JP, Guntinas-Lichius O: [The Covid-19 pandemic and otolaryngology: What it comes down to?] Laryngorhinootologie. 2020; PubMed Abstract | Publisher Full Text\n\nKandeel M, Ibrahim A, Fayez M, et al.: From SARS and MERS CoVs to SARS-CoV-2: Moving toward more biased codon usage in viral structural and nonstructural genes. J Med Virol. 2020. PubMed Abstract | Publisher Full Text\n\nKurlan R, Daragjati C, Como PG, et al.: Non-obscene complex socially inappropriate behavior in Tourette's syndrome. J Neuropsychiatry Clin Neurosci. 1996; 8(3): 311–7. PubMed Abstract | Publisher Full Text\n\nMao L, Wang M, Chen S, et al.: Neurological manifestations of hospitalized patients with Covid-19 in Wuhan, China: a retrospective case series study. MedRxiv Preprint. 2020. Publisher Full Text\n\nMartino D, Ganos C, Pringsheim TM: Tourette Syndrome and Chronic Tic Disorders: The Clinical Spectrum Beyond Tics. Int Rev Neurobiol. 2017; 134: 1461–1490. PubMed Abstract | Publisher Full Text\n\nMartino D, Zis P, Buttiglione M: The role of immune mechanisms in Tourette syndrome. Brain Res. 2015; 1617: 126–43. PubMed Abstract | Publisher Full Text\n\nMüller-Vahl KR, Kayser L, Pisarenko A, et al.: The Rage Attack Questionnaire-Revised (RAQ-R): Assessing Rage Attacks in Adults With Tourette Syndrome. Front Psychiatry. 2020; 10: 956. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPringsheim T, Okun MS, Müller-Vahl K, et al.: Practice guideline recommendations summary: Treatment of tics in people with Tourette syndrome and chronic tic disorders. Neurology. 2019; 92(19): 896–906. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRizzo R, Gulisano M, Pellico A, et al.: Tourette syndrome and comorbid conditions: a spectrum of different severities and complexities. J Child Neurol. 2014; 29(10): 1383–9. PubMed Abstract | Publisher Full Text\n\nRobertson MM, Cavanna AE: The disaster was my fault! Neurocase. 2007; 13(5): 446–51. PubMed Abstract | Publisher Full Text\n\nRobertson MM, Eapen V, Singer HS, et al.: Gilles de la Tourette syndrome. Nat Rev Dis Primers. 2017; 3: 16097. PubMed Abstract | Publisher Full Text\n\nTowers VS, Goldsmith M, Goldsmith P: Zika Virus: Patient Education Recommendations. Adv Neonatal Care. 2018; 18(5): 360–365. PubMed Abstract | Publisher Full Text\n\nvan de Griendt JM, Verdellen CW, van Dijk MK, et al.: Behavioural treatment of tics: habit reversal and exposure with response prevention. Neurosci Biobehav Rev. 2013; 37(6): 1172–7. PubMed Abstract | Publisher Full Text\n\nWan S, Xiang Y, Fang W, et al.: Clinical Features and Treatment of COVID-19 Patients in Northeast Chongqing. J Med Virol. 2020. PubMed Abstract | Publisher Full Text\n\nWright A, Rickards H, Cavanna AE: Impulse-control disorders in Gilles de la Tourette syndrome. J Neuropsychiatry Clin Neurosci. 2012; 24(1): 16–27. PubMed Abstract | Publisher Full Text\n\nWu F, Zhao S, Yu B, et al.: A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798): 265–269. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang G, Zhang J, Wang B, et al.: Analysis of clinical characteristics and laboratory findings of 95 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a retrospective analysis. Respir Res. 2020a; 21(1): 74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang JJ, Dong X, Cao YY, et al.: Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China. Allergy. 2020b. PubMed Abstract | Publisher Full Text\n\nZhang X, Cai H, Hu J, et al.: Epidemiological, clinical characteristics of cases of SARS-CoV-2 infection with abnormal imaging findings. Int J Infect Dis. 2020c. PubMed Abstract | Publisher Full Text\n\nZheng F, Liao C, Fan QH, et al.: Clinical Characteristics of Children with Coronavirus Disease 2019 in Hubei, China. Curr Med Sci. 2020a. PubMed Abstract | Publisher Full Text\n\nZheng HY, Zhang M, Yang CX, et al.: Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients. Cell Mol Immunol. 2020b. PubMed Abstract | Publisher Full Text\n\nZhu N, Zhang D, Wang W, et al.: A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "62276",
"date": "16 Apr 2020",
"name": "Kirsten R Müller-Vahl",
"expertise": [
"Reviewer Expertise Tourette syndrome",
"tic disorders",
"cannabis-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\nThis is a nicely written paper by a group of renowned European Tourette experts about GTS in times of COVID-19. The authors discuss similarities of COVID-19 and tics in GTS and outline specific problems that may result from the pandemic for this group of patients. Finally, they give concrete suggestions how to advice and treat patients with GTS and their families during the pandemic. I like this small paper and find it interesting to read, since it alerts us that the current pandemic may be much more challenging for patients with GTS compared to healthy people. I have some minor suggestions the authors may take into consideration to further improve the quality of this manuscript:\nThe authors mix the name of the virus (SARS-CoV-2) and the name of the disease caused by this virus (COVID-19). COVID-19 is simply the abbreviation of Coronavirus Disease 2019. Therefore, the authors should correct for example the heading „ Aetiology: COVID-19 (coronavirus)”. As far as I know, the correct spelling is “SARS-CoV-2” (not “SARS-Cov-2”). Also in Table 1: “Viral infection – Coronavirus 19“ please change in “SARS-CoV-2”.\n\nThe authors describe the different symptoms associated with COVID-19 including neurological complications. I suggest to include recent reports on meningitis/encephalitis (for example see: https://doi.org/10.1016/j.ijid.2020.03.062)1.\n\nCoprolalia: according to the literature it occurs in more than 10% of patients. I suggest to include a reference here too.\n\nThe authors should use the term “pandemic” throughout instead of in some cases “epidemic”.\n\nCorrect “cognitive-behavioural (CBT) training” in “cognitive-behavioural training (CBT)”.\n\nRephrase this sentence (I think the “a” must be deleted): “Touching other people is a not an uncommon complex motor tic but in the “COVID-19 climate” could also represent NOSIS (see Table 1).”\n\nTable 1: I suggest to spell “Palilalia/Palipraxia” and “Echo praxia – lalia” in the same way.\n\nI suggest to change the order: “selective serotonin reuptake inhibitors (SSRIs)” instead of “SSRIs (selective serotonin reuptake inhibitors)”.\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": "62277",
"date": "17 Apr 2020",
"name": "Andrea Cavanna",
"expertise": [
"Reviewer Expertise Behavioural neurology and neuropsychiatry."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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, informative, and well written article providing clinicians with practical advice about how to best advise and support patients with tics during these challenging times. The authors navigate through the many uncertainties of the unfolding circumstances using the best available evidence as well as their own invaluable experience, according to the ancient motto ‘scientia et conscientia’. I have little to say in terms of further improving this useful article. As the authors rightly point out, anxiety is a common comorbidity that needs to be addressed with particular care, in consideration of its recognized role as tic-exacerbating factor. A recent review article on telemedicine for hyperkinetic movement disorders covers tic disorders, and states that “the extent to which telemedicine facilitates multidisciplinary treatment needs to be further studied” (Srinivasan et al. 2020)1. It is hoped that the current circumstances and the unprecedented measures (including social distancing) are also seen as an opportunity to improve our understanding of patients’ needs. The authors might want to add their suggestions about gathering high quality information on remotely delivered clinical input.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-257
|
https://f1000research.com/articles/9-8/v1
|
09 Jan 20
|
{
"type": "Research Article",
"title": "Applying inter-rater reliability to improve consistency in classifying PhD career outcomes",
"authors": [
"C. Abigail Stayart",
"Patrick D. Brandt",
"Abigail M. Brown",
"Tamara Dahl",
"Rebekah L. Layton",
"Kimberly A. Petrie",
"Emma N. Flores-Kim",
"Christopher G. Peña",
"Cynthia N. Fuhrmann",
"Gabriela C. Monsalve",
"Patrick D. Brandt",
"Abigail M. Brown",
"Tamara Dahl",
"Rebekah L. Layton",
"Kimberly A. Petrie",
"Emma N. Flores-Kim",
"Christopher G. Peña",
"Cynthia N. Fuhrmann",
"Gabriela C. Monsalve"
],
"abstract": "Background: There has been a groundswell of national support for transparent tracking and dissemination of PhD career outcomes. In 2017, individuals from multiple institutions and professional organizations met to create the Unified Career Outcomes Taxonomy (UCOT 2017), a three-tiered taxonomy to help institutions uniformly classify career outcomes of PhD graduates. Early adopters of UCOT 2017, noted ambiguity in some categories of the career taxonomy, raising questions about its consistent application within and across institutions. Methods: To test and evaluate the consistency of UCOT 2017, we calculated inter-rater reliability across two rounds of iterative refinement of the career taxonomy, classifying over 800 PhD alumni records via nine coders. Results: We identified areas of discordance in the taxonomy, and progressively refined UCOT 2017 and an accompanying Guidance Document to improve inter-rater reliability across all three tiers of the career taxonomy. However, differing interpretations of the classifications, especially for faculty classifications in the third tier, resulted in continued discordance among the coders. We addressed this discordance with clarifying language in the Guidance Document, and proposed the addition of a flag system for identification of the title, rank, and prefix of faculty members. This labeling system provides the additional benefit of highlighting the granularity and the intersectionality of faculty job functions, while maintaining the ability to sort by - and report data on - faculty and postdoctoral trainee roles, as is required by some national and federal reporting guidelines. We provide specific crosswalk guidance for how a user may choose to incorporate our suggestions while maintaining the ability to report in accordance with UCOT 2017. Conclusions: Our findings underscore the importance of detailed guidance documents, coder training, and periodic collaborative review of career outcomes taxonomies as PhD careers evolve in the global workforce. Implications for coder-training and use of novice coders are also discussed.",
"keywords": [
"workforce development",
"higher education",
"career outcomes",
"STEM education",
"career taxonomy"
],
"content": "Introduction\n\nRecently, national conversation has highlighted the need for greater institutional transparency in reporting career outcomes of graduate and postdoctoral alumni. In addition to fulfilling institutional reporting requirements, public sharing of outcomes data provides information to prospective and current trainees about the broad career paths that leverage their PhD training. Outcomes data also informs the evolution of graduate and postdoctoral education practices and policies. In response to the current lack of career outcome visibility, organizations and funding agencies have made concerted efforts to encourage and support institutional commitment to public sharing of career outcomes data. Many groups have called for institutional transparency in career outcomes for PhD-trained scientists, including, but not limited to: the National Institutes of Health (NIH)1, the Future of Bioscience Graduate and Postdoctoral Training (FOBGAPT) conferences I & II2,3, Rescuing Biomedical Research (RBR)4,5, Future of Research (FoR)6, the UW-Madison Workshop7, the National Institute of Health Broadening Experiences in Scientific Training (BEST) Consortium8,9, the Coalition for Next Generation Life Science (NGLS Coalition)10, Council of Graduate Schools (CGS)11, the Association of American Universities (AAU)12, the Association of American Medical Colleges (AAMC)13, and the National Academy of Sciences14,15. Many institutions are now publicly sharing their alumni career outcomes data on websites and in publications8,16–19. However, a major impeding factor in this effort to share outcomes data has been the multitude of career outcomes taxonomies developed by individual institutions and agencies. The absence of a unified language for reporting the career outcomes of graduate and postdoctoral alumni perpetuates an environment in which universities, policy makers, and prospective trainees are unable to compare career outcomes on a local, regional, or national level.\n\nIn Spring 2017, 14 schools within the BEST Consortium formed a working group to design a taxonomy of career descriptions that could be used across institutions to consistently describe the career outcomes of PhD and postdoctoral alumni trained in the biological and biomedical sciences. The working group used the Science Careers myIDP20 career categories as its starting point for the taxonomy. This work was subsequently incorporated into a collaborative effort led by RBR, which convened national stakeholders, including experts in graduate training and career development from AAMC, the NIH, the AAU, and academic institutions both internal and external to the BEST Consortium. The resultant three-tiered Unified Career Outcomes Taxonomy (UCOT 2017) was developed with the aim of creating a publicly available, standardized, and valid measure that was vetted by experts at the national level. It was first released online by the BEST Consortium21, and reports of the meeting, including additional recommendations for data collection, were reported by RBR, AAMC, and BEST5,13,22,23. The process of developing UCOT 2017 and its associated recommendations was an example of cross-organizational communication, collaboration, and compromise. Several institutions, including those of the Coalition for Next Generation Life Science, have moved forward in adopting UCOT 2017 as a first step toward tracking and comparing career outcomes24.\n\nThe UCOT 2017 has three tiers of classification: Workforce Sector, Career Type, and Job Function. While the categories in each tier were deemed sufficiently broad to describe the primary career trajectories of PhDs in the biological sciences, the breadth of those definitions permitted significant room for discordant interpretation. Based on our early attempts to utilize UCOT 2017, we were concerned that the breadth of the definitions could obscure the nuances of the complex career taxonomy and result in unreliable and inconsistent application by individuals (i.e., coders) across different institutions responsible for coding the outcomes data. We were particularly concerned about coding categories in the Job Function tier which provides for a granular refinement of the specific skill sets and/or credentials required for employment within that function. Without a reliable, reproducible, and robust schema for the classification of career outcomes, cross-site comparisons would have limited validity and the value of national reporting would be diluted25. We believed it to be crucial to test the reliability of UCOT 2017 and to uncover any inconsistencies in the application of the career taxonomy by different coders. In this study, we identified taxonomic categories that resulted in low concordance (i.e., low inter-rater reliability) among coders and iteratively modified the career taxonomy until all three tiers met or exceeded reliability standards.\n\n\nMethods\n\nInstitutional Review Board (IRB) approvals for this project were obtained from each institution that provided alumni records to the study (Emory University: IRB# H13506; Vanderbilt University School of Medicine: IRB# 180315; University of North Carolina, Chapel Hill: IRB# 14-0544). Institutional consent processes for data collection and consent were reviewed separately as participating institutions of the NIH BEST consortium (variants included an electronic information sheet, inclusion of a standardized NIH BEST consortium-wide informed consent paragraph, and approval as exempt or non-human subjects research designations with no consent required). In all cases, IRB approvals included the stipulation to remove any identifiable information from participant data. In accordance with this requirement, only de-identified data are reported and all subject identification numbers have been reassigned to protect participant identities.\n\nFour coders from the BEST Consortium (PDB, TD, AMB, CAS) conducted a preliminary application of UCOT 2017 to code 2,587 graduate student alumni records from across their respective institutions (data not reported here due to IRB data-sharing limitations). This preliminary application of UCOT 2017 revealed several areas of confusion. Therefore, prior to beginning Experiment 1, this group agreed upon initial clarifications to UCOT 2017, therein generating “UCOT experimental version 1” (UCOT Exp1). Two of the group members (TD and CAS) created a Draft Guidance Document to specifically address confusing categories that were discovered during preliminary use of UCOT 2017 and were anticipated to result in discordant interpretation during Experiment 1. The Draft Guidance Document included some elaboration on how to classify faculty, entrepreneurs, and postdocs and other types of training positions.\n\nExperimental overview. Figure 1 provides an overview of our experimental design. Using sets of alumni records from three universities, our overall experiment was composed of two stages: a first round of coding using a modified version of the 2017 Unified Career Outcomes Taxonomy (UCOT Exp1), and a second round of coding (with some returning coders and some new coders) using UCOT Exp2. UCOT Exp2 is a modified version of UCOT Exp1 that was refined based on inter-rater reliability analyses and coder feedback following the first round. After each round, inter-rater reliability was calculated for each tier of the career taxonomy and taxonomic categories were identified that caused particular discordance across coders.\n\nIn Experiment 1, six coders comprising four experienced coders (blue) and two inexperienced coders (orange) classified 572 PhD alumni records from three different institutions using UCOT Exp1 and the Draft Guidance Document. After measurements of inter-rater reliability were determined, the working group convened to refine the career taxonomy definitions and guidance materials, generating experimental version 2 (UCOT Exp2) and the Revised Guidance Document. The reliability of UCOT Exp2 and the Revised Guidance Document were then assessed using a new data set of 219 PhD alumni records. In Experiment 2, coding was performed by five experienced coders (blue) and three new inexperienced coders (orange). After classification of the records, inter-rater reliability was measured. The group convened again to review the results and introduce further clarifications to the Revised Guidance Document, therein creating the Final Guidance Document.\n\nThe team involved in Experiment 1 consisted of individuals who had previous experience with the career taxonomy, including four individuals who had participated in the original BEST working group and were responsible for generating UCOT Exp1 (two were also present at the national meeting associated with developing UCOT 2017). The Experiment 1 team also included two inexperienced coders who were new to both the career taxonomy and the experiment. The team of coders involved in Experiment 2 included five experienced coders who were involved in Experiment 1 and three inexperienced coders who had no previous involvement in the project. Beyond the coders from Exp 1 who took on the task, additional coders were recruited from the NIH BEST Consortium Program Directors/Staff during NIH BEST meetings, consortium calls, and conversations. In total, nine coders participated in one or both stages of this study. At the time of this study, all nine were university administrators in professional roles focused on career development for PhD scientists, with experience ranging from six months to fifteen years in the field. Eight of the nine coders had doctoral degrees in the biological or social sciences; the team consisted of seven women and two men.\n\nThe pool of alumni records from which the experimental data sets were selected included the 2,587 graduate student alumni previously coded by Vanderbilt University, Emory University, and UNC Chapel Hill. This sample of records represented all alumni data available since the inception of each respective institutional umbrella program (e.g., Vanderbilt’s Interdisciplinary Graduate Program in Biological and Biomedical Sciences, Emory’s Graduate Division of Biological and Biomedical Sciences, and UNC’s Biological and Biomedical Sciences), excluding records that could not be verified or located. The sample was intended to establish a combined pool large enough to provide representative distribution across job categories for a robust dataset to code. Each record was composed of a unique record number, current job title, current employer, LinkedIn profile or other job-related URL, and graduation date. Postdoctoral alumni were not included in the data set due to inconsistent collection of career outcomes for this population across institutions.\n\nThe data set coded in Experiment 1 contained 572 alumni records, including 185 records from UNC Chapel Hill, 192 from Vanderbilt, and 195 from Emory. Women comprised 47% of the dataset, men comprised 28%, and 25% of the alumni records were of unknown gender. Figure 2 shows the distribution of alumni records according to year of graduation. For a detailed description of how alumni records were selected for inclusion in Experiment 1, please refer to Extended data: S4.\n\nDistribution of graduation years for all records coded in Experiment 1 using UCOT Exp1.\n\nThe data set coded in Experiment 2 contained 219 alumni records. A minimum representative sample across each Job Function category was used to select the number of records; where possible, three records per job function were chosen from each institution. Nine records per job function provide sufficient data to determine inter-rater reliability without placing an undue time burden on the coders. The Experiment 2 dataset was comprised of 49% women, 26% men, and 25% unknown. Figure 3 shows the distribution of records according to year of graduation. For a detailed description of how alumni records were selected for Experiment 2, please refer to Extended data: S5.\n\nDistribution of graduation years for all records coded in Experiment 2 using UCOT Exp2.\n\nCoders were each provided with three documents: 1) the Draft Guidance Document for how to code challenging records (generated by CAS and TD, and piloted at Emory prior to this study; included within Extended data: S2 – note that the Exp 1 version is simply the Exp 2 version without the additions annotated in yellow); 2) UCOT Exp1 with definitions (see Extended data: S2 minus the annotated additions); and 3) a Data Collection Workbook (entry-validated spreadsheet designed to collect the data; see Extended data: S7) containing the 572 records to be coded using data-validated fields for classification in each tier. Each record was composed of a unique record number, job title, current employer, LinkedIn profile or other job-related URL, and graduation date. Each record in the workbook was coded by selecting from drop-down menus of categories in each taxonomic tier (Workforce Sector, Career Type, and Job Function). For each record, coders were also prompted to indicate (yes/no) whether they had accessed the provided LinkedIn profile. In order to combine the data, since each column/variable used identical headers to indicate which sector was being coded, a rater number was added to each to identify which source worksheet it had originated (e.g., Sector 1_Rater 1, Type_Rater 1, Function_Rater 1; Sector 1_Rater 2, and so forth). Data was collected in separate MS Excel spreadsheets and uploaded into the SPSS software package (Version 24) in preparation for data analysis.\n\nTo examine categorical consistency across coders, we used Krippendorff's Alpha26, which estimates the level of agreement (inter-rater reliability) among coders. Parameters of acceptable reliability using Krippendorff's alpha start at a lower bound of 0.67 (for a review, see 27; estimates range from 0.67 – 0.80), with 0.70 being the convergent recommendation used to define reliability among coders (25 gives the caveat that 0.67–0.80 should be interpreted with caution). Hence, we set 0.70 as our comparison point for an acceptable measure of reliability and replicability of the taxonomies tested in this work. Inter-rater reliability was calculated using SPSS, with an amended macro designed for Krippendorff's alpha28.\n\nIn addition to calculating inter-rater reliability, we employed two methods -- average number of unique categories and percent discordance -- to identify discordance between coders. Both methods share a common starting point, which was to determine the Workforce Sector, Career Type, and Job Function that was most commonly chosen (the mode) by the coders for every record. The mode was then designated as the presumed “correct” answer. To calculate the average number of unique categories per tier for each record (columns A and C in Extended data: S6), we counted the number of unique categories that coders applied to the given record. We then calculated the average number of unique categories across all records in each tier (as defined by the most popular choice). Percent discordance was measured by determining, for every record, how many coders did not choose the same category as the mode. Subsequently, that number was divided by the total number of coders and multiplied by 100 to give a percentage (columns B and D of Extended data: S6).\n\nFollowing Experiment 1, the group of six coders met twice by phone to review the data, discuss sources of discordance (see Figure 1 and Methods), and ways to improve consistency between coders. These meetings resulted in UCOT Exp2, which was tested in Experiment 2, and a new version of the Guidance Document (“Revised Guidance Document”) that was significantly expanded and refined from the draft version used in Experiment 1. Modifications within the Revised Guidance Document included: a) a formal introduction to the career taxonomy classification system and recommendations for implementation; b) a set of frequently asked questions (FAQs); c) the taxonomic categories with refined definitions; and d) a reference table containing the list of Carnegie Classifications of Academic Institutions to aid in the classification of Career Types as “Primarily Research” or “Primarily Teaching”, if needed (see Discussion). The guidance FAQs addressed and provided proscriptive clarification for how to code types of jobs that had been identified as problematic in Experiment 1, as well as explanations for the logic behind the recommendations. Examples included how to:\n\nCode training positions (e.g., Fellows, Scholars, Residents, Interns)\n\nDistinguish between “Principal Investigators” and “Research Staff”\n\nDefine “Entrepreneur”\n\nAssign a primary job function for individuals with multiple roles\n\nDifferentiate “For-Profit” from “Nonprofit” entities\n\nDifferentiate “Academic” from “Nonprofit” hospitals\n\nResolve assignment of faculty members to Career Type “Primarily Research” or “Primarily Teaching”\n\nImplement the Faculty Flag system to further classify alumni in faculty roles (see Discussion).\n\nA new set of 219 alumni records was selected and coded using UCOT Exp2 and the Revised Guidance Document (see annotated changes in Extended data: S2). The coding team included five experienced coders from Experiment 1 and three inexperienced coders who were new to the project. New coders were intentionally recruited to participate in Experiment 2 to explore whether the Revised Guidance Document and definitions of updated taxonomic categories were sufficient to increase the reliability of coding by an inexperienced user.\n\nIn Experiment 2, coders were provided with: 1) the Revised Guidance Document; 2) UCOT Exp2 (Extended data: S1); and 3) a fresh Data Collection Workbook containing 219 alumni records to be coded using data-validated fields for classification in each tier. The Data Collection Workbook used in Experiment 2 included the Carnegie Classification of each academic employer29 and three new open-answer coding fields, including: 1) drop-down menu related to collecting Faculty Flag data (if relevant, as discussed below); 2) prompt to note whether additional sources had been accessed (e.g., institutional website, personal lab website, etc.,); 3) prompt to indicate how much time was spent coding the record in minutes.\n\nUpon completion of the coding phase of Experiment 2, inter-rater reliability analyses were performed on the results and the group of eight coders met by phone to identify where ambiguity remained in UCOT Exp2. Two areas of confusion were discussed (specifically, classifying the Workforce Sector of K-12 school teachers and the Job Function of entrepreneurs) and the Revised Guidance Document was modified (creating the Final Guidance Document) to provide more detail on how to code alumni in these career paths; no changes were made to UCOT Exp2. Extended data: S1 and S3 contain the UCOT Exp2 and the Final Guidance Document, respectively.\n\nWe provide summaries of the tiers, categories, and definitions used in Experiment 2 in Table 1–Table 3.\n\nAn individual’s Workforce Sector generally reflects the type of company or institution where they are employed. The unabridged version of UCOT Exp2 including example titles is available in Extended data: S1.\n\nAn individual’s Career Type should reflect the general content of their work. The unabridged version of UCOT Exp2 including example titles is available in Extended data: S1.\n\nAn individual’s Job Function is defined by specific skill sets and/or credentials required for employment in that function. The unabridged version of UCOT Exp2 (Extended data: S1) includes Job Function, Definition, Coding Clarifications, Job Title Examples, and Common Coding Scheme.\n\n\nResults\n\nIn this study, we tested the consistency and applicability of the Unified Career Outcomes Taxonomy (UCOT 2017;23) and a guidance document that we generated for this study. This was done by assessing the inter-rater reliability of classifications generated by multiple coders using the career taxonomy. Inter-rater reliability was evaluated using three measures: Krippendorff’s Alpha, number of unique category classifications, and percent discordance. We repeated the experiment with iterative refinements to the career taxonomy and guidance document until differences across coders were sufficiently reliable based on published thresholds for Krippendorff’s Alpha.\n\nBased on our preliminary application of UCOT 2017 (data not reported here), we initiated Experiment 1 by modifying UCOT 2017 to clarify several of the definitions of taxonomic categories, therein generating UCOT Exp1. This version was used to code 572 records. Using a Krippendorff's Alpha of 0.70 as the minimal threshold to define reliability among six coders (see Methods;25,26), we observed that the Workforce Sector tier (0.83 agreement) and the Career Type tier (0.70 agreement) met acceptable parameters of reliability (Figure 4). However, the measured reliability for the Job Functions tier (0.62 agreement) was below admissible levels (Figure 4).\n\nWorkforce Sector, Career Type, and Job Function were coded by six coders in Experiment 1 (black bars) and eight coders in Experiment 2 (gray bars). The 0.7 threshold is indicated by the horizontal dotted line. Experiment 2 results for every category are higher than Experiment 1. Coders used 6 categories for Workforce Sector, 6 categories for Career Type, and 26 (Experiment 1) or 23 (Experiment 2) categories for Job Function.\n\nThe six coders in Experiment 1 had six-way agreement on 77% of all records within Workforce Sector, 55% of all records within Career Type, and 36% of all records within Job Function. Furthermore, we reached five-way agreement on 95% of records for Workforce Sector, 95% of records for Career Type, and 73% of records for Job Function, suggesting that, in many cases, only one person interpreted a definition differently than the rest of the group.\n\nTo identify hot spots of discordance, we ranked the records by number of unique categories utilized by the coders and looked for patterns among records that had high disagreement (an illustration of the discordance is provided in Extended data: S6). Within the Career Type tier, we identified discordance in three of the six categories, specifically, application of the “Further Training” category and in differentiating “Primarily Research” and “Primarily Teaching” faculty positions. Within the Job Function tier, we discovered that seven of twenty-three categories presented high levels of discordance, including: “Faculty- Non-Tenure track”; “Clinical Services”; “Sales and Marketing”; “Group Leader”; “Business Development, Consulting, and Strategic Alliances”; “Clinical Research Management”; and “Administration”.\n\nWhile we hypothesized that most instances of discordance could be addressed by providing additional examples in the Draft Guidance Document or by making minor clarifications in taxonomic definitions, we also hypothesized that two structural changes to UCOT Exp1 would improve reliability. To address the discordance in the coding of the five faculty Job Functions in UCOT Exp1 (“Adjunct/part-time teaching staff”, “Faculty: non-tenure track”, “Faculty: tenured/tenure track”, “Faculty: tenure track unclear or not applicable”, and “Instructor/full-time teaching staff”), the group agreed to test an alternative approach to capturing faculty data, originally developed by the Biomedical Research Education and Training office (BRET) at Vanderbilt (KP and AB, unpublished study). Specifically, in addition to classifying each record by the three primary taxonomic tiers, the record is also examined for any indication in the job title that the individual holds a faculty designation at their employing institution. If so, the record is ‘flagged’ as faculty and additional information is collected, including title (e.g., professor, instructor, lecturer), rank (e.g., assistant, associate), and prefix (e.g., adjunct, research, teaching). This strategy for capturing faculty status permitted us to combine the five faculty-related Job Functions in UCOT 2017 and UCOT Exp1 into just two Job Functions in UCOT Exp2: “Principal Investigator or Group Leader” and “Teaching Faculty or Staff.” Examples of the Faculty Flag schema with sample data are provided in the Final Guidance Document (Extended data: S3). The second structural change was introduced into the Career Type tier by adding an “Other” category; this permitted coders to more accurately capture alumni for whom some information is known, thereby obviating the default classification as “Unknown”. Incorporation of these changes resulted in the UCOT Exp2, which was then used in Experiment 2 by a second group of coders to code a new dataset of 219 records (see Methods). Among the eight coders for UCOT Exp2 we calculated Krippendorf’s alpha for the Workforce Sector tier (0.90 agreement), the Career Type tier (0.73), and the Job Functions tier (0.69 agreement). Notably, we observed improved inter-rater reliability across all three tiers in Experiment 2 (Figure 4).\n\nWe considered that the elevated reliability achieved in Experiment 2 using UCOT Exp2 may have resulted from the coders’ greater familiarity with the career taxonomy, developed through Experiment 1 and subsequent conversations about sources of discordance. To explore this possibility, reliability was calculated separately for experienced and inexperienced coders in Experiment 2. Although experienced coders performed slightly better, inexperienced coders did not lag far behind (Figure 5). Experienced coders using UCOT Exp2 met standards for acceptable reliability for each tier, including Krippendorf’s alpha for the Workforce Sector tier (0.93 agreement), the Career Type tier (0.78), and the Job Functions tier (0.70 agreement).\n\nIn Experiment 2, which included experienced and inexperienced coders, the results are similar for experienced (n=5, striped bars) and inexperienced coders (n=3, white bars). Of note, experienced coders met acceptable standards of reliability for each tier. The 0.7 threshold is indicated by the horizontal dotted line.\n\nThe introduction of inexperienced coders to Experiment 2 also permitted us to explore whether the amount of time spent coding each record significantly differed between inexperienced and experienced coders. Accordingly, in Experiment 2 all coders were prompted to record how much time was spent coding each record. The total time for experienced coders ranged from 213 to 540 minutes (average 328 minutes) and 246 to 545 minutes for inexperienced coders (average 429 minutes). The overall average time to classify the second round of 219 records was 366 minutes, which equates to about 1 minute, 40 seconds per record.\n\n\nDiscussion\n\nIn this study, we tested the inter-rater reliability of multiple coders categorizing the records of PhD alumni using a collaboratively-established, three-tier career outcomes taxonomy, UCOT 2017. Through two rounds of refinement (Experiment 1 and Experiment 2) of UCOT 2017 and a companion guidance document, we achieved satisfactory inter-rater reliability across all three tiers of the career taxonomy. We ascribe the majority of the improvement in reliability during Experiment 2 (Figure 4) to the provision of a detailed Revised Guidance Document, which was largely informed by the classification of 2500+ alumni records prior to the initiation of experiments reported here. For the experiments in this study, we intentionally enriched the alumni data set of 800 records with less common Job Functions to ensure full coverage of the career taxonomy, with the hope that we would encounter uncommon types of employment that were poorly defined or described in the taxonomy. While there are likely to be unique forms of employment that we did not anticipate or encounter in our datasets (and therefore are not addressed in the Final Guidance Document), we are confident that we obtained full coverage of the most common current career outcomes for PhDs in the biosciences and that the Final Guidance Document (Extended data: S3) provides sufficient instruction to reliably code these career outcomes. The strength of UCOT 2017 is evident in its overall performance in our study, given the increased representation of challenging job titles in our alumni samples.\n\nOur experiments indicate that institutions should consider the value of training and retaining experienced coders year after year; however, the use of inexperienced coders should not preclude an institution from starting to classify alumni career outcomes (Figure 5). As coders are first trained, we recommend that institutions internally measure and evaluate inter-rater reliability on sample records. To that end, institutions or academic divisions may find that it is most efficient to designate a small, centralized team to code all alumni records, as opposed to approaching classification in a more decentralized manner that involves many inexperienced coders. We also recommend that groups aiming to use a career taxonomy to compare across institutions meet to develop a detailed guidance document to ensure common interpretation of categorical definitions.\n\nIn the process of identifying discordances through this study, we adapted the UCOT classification system and refined our corresponding guidance document to improve reliability. These changes are detailed below. See also Extended data: S2, which contains annotations for each change comparing UCOT Exp2 with the original UCOT 2017 (this also allows for ease of comparison and/or cross-walking one’s data between versions as needed).\n\nTier 1: Workforce Sector. In the Workforce Sector tier, we defined Academia as “any post-secondary academic institution where training occurs, including colleges, universities, some medical centers, or freestanding research institutions.” We found discordance among coders when assigning K-12 teaching jobs to Workforce Sector and, as a result, we concluded that K-12 schools would be coded as Government, Nonprofit, or For-Profit based on publicly-available information about that school. For example, public K-12 schools are most properly classified in the Government sector, and private schools are either For-Profit or Nonprofit (see Extended data: S3).\n\nTier 2: Career Type. In the Career Type tier of UCOT Exp2 we added the term ‘unemployed’ to the existing category “Unknown,” renaming this category “Unemployed/Unknown.” In the absence of this category, individuals who are not currently employed but who are known to be searching for a job or intend to return to the workforce could only be classified as Career Type “Unknown,” thereby losing information that may actually exist about their current employment status. Now, by classifying them as “Unemployed/Unknown” at the Career Type tier and combining that with a new Job Function category, “Unemployed or Seeking Employment” (described below), we are able to capture current status of individuals who have described themselves as, for example, family caregivers or on the job market.\n\nSecond, classification of faculty as “Primarily Research” versus “Primarily Teaching” is complicated by the multi-functional roles played by many faculty members and by the fact that a large proportion of academic faculty balance both research and teaching. We instructed the coders to use available public resources to determine in which capacity the faculty member is likely to spend most of their time. Examples of this type of data included but are not limited to: frequency and recency of peer-reviewed research articles (e.g., publication record on PubMed); information related to the number of courses that an individual taught in a given academic year; and, supervising the research of doctoral students or postdoctoral scholars.\n\nIf no illuminating data could be found to facilitate classification of a given faculty member as “Primarily Research” or “Primarily Teaching”, we stipulated that a coder should use the Carnegie Classification29 of the employing institution to classify Career Type. For example, if the individual was a faculty member at an academic institution with a Carnegie Classification of 15 or 25 (“highest research activity”), the coder should categorize the individual as “Primarily Research”. Faculty at institutions in any other Carnegie Classification should be categorized as “Primarily Teaching.” (See further examples of this coding strategy in the Final Guidance Document, Extended data: S3). We urged coders to gather data from online sources listed above and to avoid using the Carnegie Classification strategy due to its potential inaccuracy. For example, defaulting to the Carnegie Classification would misrepresent some faculty as “Primarily Research” who are actually “Primarily Teaching” because many institutions with Carnegie Classification 15 or 25 hire faculty members to serve in primarily teaching roles such as instructors or teaching professors. This default strategy could also underreport the research responsibilities held by many faculty members at institutions with Carnegie Classification other than 15 or 25.\n\nThird, we addressed the classification of postdoctoral scientists in the Career Type tier. In UCOT 2017, postdoctoral scientists are classified as “Further Training or Education” at the Career Type tier, followed by “Postdoctoral” at the Job Function tier, therein losing the specific nature of their postdoctoral work. For example, if the individual is engaged in a postdoctoral training program with a significant teaching focus, the UCOT 2017 taxonomy does not capture the teaching emphasis of their training. As a consequence, one could lose opportunities to uncover distinct shifts among PhD outcomes, such as identifying longitudinal trends in the types of postdoctoral training experiences pursued by our trainees (e.g., a decrease in number of research-focused postdocs accompanied by a rise in teaching-focused or government policy-focused postdoctoral positions). Facilitated by the possibility that their postdoctoral training status could be captured in the Job Function tier, we instructed coders to classify postdocs as “Primarily Research” or “Primarily Teaching” instead of as “Further Training or Education”6,30,31. This strategy allowed us to collect additional detail of the nature of their postdoctoral experience and greater resolution pertaining to a population that the academic community would like to track and analyze more precisely. By using the more detailed classification during data collection and coding, it is easy to later choose to report postdocs consistently with UCOT 2017 or UCOT Exp2, as desired. Alternative methods which would satisfy both reporting options are discussed further (below).\n\nSome institutions using UCOT 2017 are collecting and reporting data for tiers 1 and 2 (Workforce Sector and Career Type), but not for tier 3 (Job Function). This raises a caveat to our proposed solution for categorizing postdoctoral scientists: in the instance where an institution is not collecting the Job Function tier, we agree that postdocs should be classified as “Further Training or Education” in Career Type (tier 2) to highlight the temporary nature of the postdoctoral position. Otherwise, the institution will undercount the number of graduates going on to postdoctoral positions.\n\nOur proposed solution is one of many potential solutions to this challenge of collecting more nuanced detail about postdoctoral outcomes. Alternate strategies could include:\n\ncreating additional Job Functions to capture different types of postdoctoral positions (e.g., “Postdoctoral: Research” and “Postdoctoral: Teaching”);\n\nimplementing a “Postdoc Flag” to capture the focus of the postdoctoral position (e.g., research, teaching, policy, etc.). In the latter case, the individual would be classified as Career Type “Further Education or Training” (as is currently recommended in UCOT 2017), followed by Job Function “Postdoctoral,” and an additional data collection field would prompt the coder to select the focus of the postdoctoral position (research, teaching, policy, etc.); or,\n\nremoving postdoctoral/further education/training categories from Career Type and Job Function tiers, then introduce a “Training Flag” (denoting “Student” or “Postdoctoral”) in conjunction with classifying them in a category that is most similar to their type of work.\n\nRegardless of strategy, we recommend erring on the side of collecting as much data as possible from the beginning of the process, as it is more efficient (e.g., less time- and resource-intensive) to collect data points all at once than it is to go back to gather additional data as an afterthought.\n\nAs a corollary, it may be appropriate for the “Further Training or Education” category within the Career Type tier to be reserved explicitly for specialized training experiences distinct from the traditional research or teaching postdoc; examples include science policy fellowships (e.g., American Association for the Advancement of Science (AAAS) Science and Technology Policy Fellowship) or academic programs that will lead to an additional degree or certification (e.g., law school, MD/PhD students in second stage of training). When this strategy is used, we suggest that the focus of the further training can be captured at the Job Function tier. For example, a student in law school would be classified as “Academia → Further Training or Education → Intellectual Property and Law.” Similarly, an AAAS Fellow would be classified as “Nonprofit → Further Training or Education → Science Policy and Government.” The Final Guidance Document (Extended data: S3) includes additional examples of how an institution might choose to apply Job Function categories to postgraduate and postdoctoral training programs.\n\nTier 3: Job Function. Through iterative experimental modifications, we identified three clarifications to definitions in the Job Function tier and one larger structural modification which increased concordance among coders. Below we summarize these modifications; detailed definitions and annotated changes are provided in Extended data: S2.\n\nFirst, we added a new Job Function category “Unemployed or Seeking Employment” to more accurately classify individuals for whom some information is known about their employment status (e.g., individuals who are known to be family care providers or seeking employment). In UCOT 2017, all of these individuals would be classified as Job Function “Unknown.” Capturing data about these individuals will help institutions track the individuals who are intentionally not employed (family care providers) and those who are actively looking for employment.\n\nSecond, we found that our group faced significant challenge in distinguishing between the Job Functions “Administration” and “Business Development, Consulting, and Strategic Alliances”. Upon further discussion, we concluded that discordance resulted from specific job titles such as “Chief Financial Officer” or “Academic Dean” that relate to the administration of a company/institution (and therefore might be classified as “Administration”) yet entail significantly more responsibility and a different skill set than a departmental administrator or program manager. We suggest that these Job Functions could be differentiated by the primary focus of their work (defined as 50% or more of an individual’s time spent on these duties): strictly administrative roles spend most of their time in duties that are internal to the organization, while business development roles are generally higher-level positions that have externally-facing responsibilities.\n\nUnder this revised definition, a dean of the biological sciences division would be classified as “Academia → Science-Related → Business Development, Consulting, and Strategic Alliances,” while a departmental administrator would likely be classified as “Academia → Science-Related → Administration.”\n\nExtrapolating to other Workforce Sectors, a project review manager at a For-Profit industry company would be classified as “For-Profit → Science-Related → Administration,” while the director of medical affairs at the same for-profit industry research company would be classified as “For-Profit → Science-Related → Business Development, Consulting, and Strategic Alliances.” In the Government Sector, a grant administrator at National Institutes of Health would likely be classified as “Government → Science-Related → Administration,” and the director of the National Institutes of Health would be classified as “Government → Science-Related → Business Development, Consulting, and Strategic Alliances.” We recognize that the individual tasked with classifying alumni records may not be able to distinguish the full spectrum of duties involved in a specific job and may feel ill-equipped to categorize these individuals. We encourage coders to use public data sources to their fullest extent, accepting that, as in all projects that are based in subjective assessment, there will be some degree of error introduced by interpretation.\n\nThird, we identified discordance generated by job titles that included ‘medical science liaison’, which UCOT 2017 classifies as a “Sales and Marketing” Job Function. We discovered that, even within our group of career development professionals, there were different understandings of what a medical science liaison actually does on a day-to-day basis; this is an example of a job that has evolved significantly over the past decade and can vary greatly depending on the employer. After significant discussion and outreach to individuals who hold the ‘medical science liaison’ job title, we concluded that, by default, these individuals should be classified in the “Technical Support and Product Development” Job Function. We recognize that there may be exceptions to this rule and encourage coders to obtain as much publicly-available information as possible about the individual’s actual duties to make the most informed categorization for each unique individual.\n\nA significant modification we made that dramatically increased inter-rater reliability and the ease of coding faculty members relates to the classification of academic faculty and teaching staff. In UCOT 2017, academic faculty are classified according to their tenure status: “Faculty: Tenure track”, “Faculty: Non-tenure track”, or “Faculty: Track unclear or not applicable.” During preliminary use of UCOT 2017 (not reported here), multiple classification strategies emerged among the coders. Some coders assumed tenure status based on the institution’s Carnegie Classification (R1, R2, etc.) and, in the absence of an ‘adjunct’ preface in their faculty title, classified nearly all faculty positions as “Faculty: Tenure track.” Other coders concluded that a tenure-track assignment could not be made without direct confirmation from the individual, given that many institutions appoint non-tenure track assistant professors or have eliminated the traditional tenure structure altogether19. We anticipated that our larger-scale experimental application of UCOT 2017 would reveal the same inconsistencies in application of these categories and thus, in the Draft Guidance Document provided to coders prior to initiating Experiment 1 (see Figure 1 for experimental procedure), we included the specific instruction not to code faculty as “Faculty: Tenure-track” unless the coder was absolutely certain that the individual held a traditional tenure track faculty appointment. This clarification of instruction resulted in zero alumni records being classified as “Faculty: Tenure-track” category during Experiment 1 (Extended data: S6). Even so, we still observed high discordance in application of the other faculty-related Job Functions (“Faculty: Non-tenure track” and “Faculty: Track unclear or not applicable”) and the determination of Career Type as “Primarily Research” or “Primarily Teaching.”\n\nIn light of the inability of multiple career development professionals to consistently and accurately assess the tenure status of an individual based on their job title and home institution, as well as changing attitudes about academic tenure32, we suggest that individuals with academic titles, such as professor, instructor, adjunct professor, research associate professor, be classified under “Principal Investigator or Group Leader”, “Research Staff or Technical Director”, or “Teaching Faculty or Staff”, based on information sourced from an individual’s public web presence (PubMed, LinkedIn, institutional or personal web pages). In order to permit finer resolution of the faculty population, we recommend that each faculty record be identified by a Faculty Flag and further notated with their faculty title, rank, and prefix (see Methods and Extended data: S3). In our experiment, adding the Faculty Flag to UCOT Exp2 greatly reduced discordance in the classification of faculty positions (Extended data: S6).\n\nImplementation of the Faculty Flag in UCOT Exp2 yielded multiple benefits. Five previous faculty Job Functions (“Adjunct/part-time teaching staff”, “Faculty: non-tenure track”, “Faculty: tenured/tenure track”, “Faculty: tenure track unclear or not applicable”, and “Instructor/full-time teaching staff”) were reduced to two (“Principal Investigator” and “Teaching Faculty or Staff”), resulting in a more simplified career taxonomy. Furthermore, our coders captured and categorized more information from alumni records than just a presumed tenure status. Specifically, this strategy allowed coders to use the Job Function category to describe the functional duties that comprised the majority of a faculty member’s time, without undercounting or misreporting their faculty appointment. For example, the Faculty Flag permitted coders to classify faculty who hold multiple academic titles, such as deans and directors, as having a “Business Development, Consulting, and Strategic Alliances” (e.g., Provost, University Chancellor/President) or “Administrative” (e.g., Chair, Dean) Job Function, respectively, while simultaneously recording their faculty status. This is intended to enhance the career taxonomy’s ability to identify faculty with broad leadership roles (externally or internally, respectively), rather than failing to recognize the impactful nature of their work within the academy and beyond. We acknowledge applying the category “Business Development, Consulting, and Strategic Alliances” to faculty and other institutional leaders may be confusing at first glance. Hence future revisions may consider using the same words reordered to lead with Strategic Alliances (e.g., “Strategic Alliances, Business Development, and Consulting”), since this function may be more readily recognizable in reflecting individuals who hold multiple roles. For additional examples of the versatility of the Faculty Flag approach, please see Extended data: S3.\n\nFinally, removing the focus on tenure from the faculty Job Function categories allowed us to identify similar roles across Workforce Sectors. Specifically, the definition “a role that involves leading a research team in any research environment where the individual is clearly responsible for funding and managing a research group” equally describes a principal investigator at an academic institution and a group leader at an industry research company. Therefore, we combined the two into a single Job Function category: “Group Leader or Principal Investigator.” This combination can easily be re-sorted into faculty and non-faculty subgroups as needed more detailed demographic analysis, while structurally reflecting the similar roles played in these two roles. To disambiguate the combination, the Workforce Sector classification (based on the individual’s employer) will clarify whether the individual performs this function in an Academic, For-Profit, Nonprofit, or Government environment. By recognizing the homology that exists between principal investigators in academia and group leaders in other sectors, UCOT Exp2 underscores the suitability, relevance, and contributions of PhD-trained scientists across the workforce.\n\nWe recognize that the collection and classification of records at the granular resolution of Job Function may seem burdensome or unnecessary to some institutions. Also, while Job Function data is extremely useful in developing career support services internal to the institution, it may be less relevant to national reporting standards. Indeed, the NGLS institutions have agreed to publish the Workforce Sector and Career Type tiers and are not requiring public reporting of the Job Function tier. However, we strongly recommend the collection of Job Function data, even if it is not publicly disseminated, since the data can have great value at the local, intra-institutional, and national levels. More than Work Sector or Career Type, Job Function data provides valuable insight into the types of skills sets used by PhDs and can inform the ongoing development of graduate and postdoctoral training support services to address changing needs in the scientific workforce. Job Function data also serves the current and prospective trainees who can gain insight into the breadth of career paths available to them and then leverage alumni who have pursued specific careers to make more informed career decisions for themselves. PhD training endows individuals with a versatile skill set, as demonstrated by PhDs who become serial entrepreneurs, government policy advisors, science educators, and public communicators, all contributing to society in ways that can be claimed and espoused by the institutions that prepared them to be successful in those ways. Collecting and publicly disseminating the breadth of career paths that PhDs pursue is essential for institutions, policy makers, and the public to assess the impact of their investment in the PhD training enterprise.\n\nIdeally, adoption of a single career taxonomy across all institutions would enable inter-institutional analyses of alumni career outcomes. However, establishing a static and universally-acceptable career outcomes taxonomy across institutions will be challenging and may not be realistic. Our results underscore that clarifications to UCOT 2017 will be needed as the workforce evolves. Additionally, individual institutions or disciplines may find that some taxonomic categories are not fully applicable in their environment; others may choose to alter definitions or create additional job functions to better suit their needs. For any changes that a group introduces to UCOT 2017, we urge institutions to clearly and prominently annotate these definitional differences when displaying the data such that it will be clear when data are comparable or not, hence avoiding cross-institutional data comparisons that are inappropriate. Furthermore, we encourage the community to come together periodically to discuss challenging cases, consider recommended changes, and test and refine inter-rater reliability for new definitions. This will be necessary if we are to sustain a career outcomes taxonomy that enables merged and comparative analyses across institutions. In order to serve both the purpose of evolving the career taxonomy, while simultaneously maintaining the ability to compare data, our guidance document includes “cross-walk” instructions for institutions to easily use their preferred classification choice, while also reporting national outcomes using consistent definitions (e.g., CNGLS current reporting requirements). This can easily be accomplished using the same data set, and very minimal additions to your data collection system. Several groups are already employing variants of UCOT Exp2 as the basis of research projects, having made local adaptations that serve their unique tracking questions17,18; personal communications from S.S. Kleppner, PhD, Associate Dean for Postdoctoral Affairs, Stanford University, on 09/22/2019, W.T. McCormack, Professor, Pathology and Immunology & Laboratory Medicine, University of Florida, 10/22/2019; T.L. Dennis, COM, Assistant Director for Postdoctoral Affairs, J.K. McClarin, Office of Academic Career Development, & D.F. Zellers, PhD, University of Pittsburgh on 10/21/2019]. We propose that the addition of the flag system in UCOT Exp2 provides an adaptable approach that permits institutions to tailor the career taxonomy in order to accommodate a variety of reporting needs.\n\nAnother perceived limitation of UCOT 2017 is its specificity to the biological sciences. Interestingly, early application of UCOT 2017 suggests that it can easily be adapted for other disciplines. For example, cross-disciplinary implementation of UCOT 2017 at Wayne State University identified that, after replacing any occurrence of the word ‘science’ in the career taxonomy with the word ‘discipline,’ the existing Career Types and Job Functions were sufficient to classify career outcomes across disciplines (A. Mathur, personal communication on 06/22/2018;33). In practice, in the Career Type tier, changing the category “Science-Related” to “Discipline-Related” or, in Job Functions, changing the category “Science Communications” to “Discipline-Related Communications” made the career taxonomy sufficient for cross-disciplinary implementation, with the single exception of adding the category “Author” to the Job Function tier. As the number of institutions that have adopted UCOT 2017 increases, we encourage continued collaboration across disciplines to further explore the versatility and applicability of the tool.\n\nOne criticism of the current taxonomic nomenclature is the use of “Not Related to Science” and “Science-Related” in the classification of Career Type due to the negative connotation associated with a career that is not directly related to one’s training. Specifically, jobs categorized as “Not Related to Science” may be interpreted as unattractive, unusual, or not requiring or valuing PhD training -- none of which may be true. Negative social consequences of the language used to mark a category as ‘other’ have been broadly explored in studies of gender, race, and even mathematical skill34. This taxonomy’s definition of “Not Related to Science” is “a career that is not directly relevant to the conduct of scientific research”, and, while we may agree that there are job types that fit that definition, one should not assume that the scientific skills and/or knowledge developed during research training are not used in those roles. For example, “children’s book author” may seem to be “not related to science,” however the author may publish books with science themes based in their scientific training. Because detailed information on the nature of work will not be available in all cases, we accept that coders will need to make educated guesses for classification between \"Not Related to Science\" and other Career Types. We hope that, as scientists innovate new roles for themselves throughout the workforce, the predominant negative valuation of careers ‘not related to science’ will be challenged. We underscore that future iterations of UCOT 2017 could explore better ways to identify and classify the diverse career trajectories of PhD alumni without using “othered” terminology.\n\nA major strength of UCOT 2017 is that it was collaboratively developed by a diverse group of scientists, including academic faculty, institutional leaders, and career development professionals. The diversity of this group and the group's collective knowledge of job functions across sectors lends itself to the validity of this instrument. However, most of the creators were employed or had strong roots in academia, which likely affected the taxonomy’s design and interpretation. Therefore, we must consider that biases were potentially introduced into the career taxonomy due to the academic background of the taxonomy developers. To ensure inclusive representation from all career fields, we recommend that future discussion of changes to UCOT 2017 include employers in sectors outside the academy.\n\nIt has not escaped our attention that one shortcoming of all versions of UCOT 2017, discussed at length during its generation and refinement over the past three years, is its inability to sufficiently represent situations where an individual balances multiple job functions. For example, there is currently no way to notate the balance of research and teaching that is expected of faculty members who might otherwise be categorized as Career Type “Primarily Teaching” and Job Function “Teaching Faculty or Staff.” Physician scientists and other cross-functional roles provide the same challenge to accurate coding of their position. We anticipate that the ‘flag’ approach could be used in additional ways by institutions that want to collect more nuanced outcome data. For example, one potential solution to this challenge could involve further elaboration of the Faculty Flag fields to include additional ‘yes/no’ drop-down prompts that investigate the contractual expectations of their appointment, e.g., research, teaching, clinical service, and/or service expectation. We recommend that the outcomes tracking community consider whether and how to address this currently unaddressed challenge.\n\nA recognized limitation of our study is that the coding group in our study consisted entirely of career professionals in graduate education; in reality, not all institutions will have a PhD-trained career professional available to do this, nor would the individual in this role necessarily have the time to code and regularly maintain an up-to-date career outcomes database. Consequently, the career taxonomy must be tested by novice coders outside of graduate career and professional development. Initial steps in this direction have been initiated, including at two institutions to date (Stanford University and the University of Chicago). Initial results suggest that, indeed, novices without graduate career development expertise training will need additional training and systems in place to ensure high-quality data coding (e.g., UCSF’s systematic data verification process24; Stanford University’s multi-user reliability check, currently in pilot phase). Further refinement and testing will be needed to ensure reproducible and consistent results across novice coders with different expertise and training backgrounds. In many cases graduate assistants (especially doctoral candidates with an interest in career and professional development or higher education administration careers) could provide a readily accessible solution, leveraging their personal experience in graduate training along with the motivation and interest to learn such taxonomic distinctions. This type of testing and implementation of verification systems will be essential to in the practical application of the career taxonomy as it is deployed nationally.\n\n\nConclusion and future directions\n\nGathering and reporting longitudinal alumni career outcome data is an important and weighty task, but one that is within reach for all graduate and postdoctoral programs. Data classified via a well-defined career taxonomy can meet the practical needs of graduate students and postdoctoral scholars who are exploring career paths, research institutions that are strategizing for the future of research and education, funding agencies that are investing in the training and growth of the national scientific workforce, and other relevant stakeholders. Having a well-defined and validated career taxonomy to code alumni outcome data will make collecting and understanding career outcomes for PhDs maximally useful internally, cross-institutionally, and nationally. We believe that UCOT 2017, in its original state as made available at the conclusion of the RBR working group, approaches fulfillment of that goal. However, we identified -- first informally via discussion, then rigorously via this study -- that there are several parts of the career taxonomy that permit variable interpretation and that we were unable to apply it reliably. Through an iterative and scientifically rigorous process, we tested whether the career taxonomy met basic standards for inter-rater reliability, and developed an example revision of the taxonomy (UCOT Exp2) which improved inter-rater reliability for all three tiers. We share UCOT Exp2 and the Final Guidance Document to demonstrate that relatively small alterations can improve functional application of the career taxonomy. For those institutions that may be attracted to the additional types of data collected through UCOT Exp2 but want to maintain the option to publicly display data in accordance with UCOT 2017, closer inspection of the differences will reveal that there are simple ways to convert between them. Regardless of taxonomic categories, we encourage institutions to include a clear description of definitions used to classify the records when publishing outcomes data publicly on institutional websites.\n\nSustaining a unified career taxonomy across many institutions will require working together as a community in an ongoing fashion. Any career taxonomy adopted by an institution or group of institutions will require periodic updating as the career landscape evolves. We recommend that the reliability of a career taxonomy be periodically revisited, tested, and updated accordingly. Testing, maintaining, and evolving any career outcomes taxonomy will benefit from the collective expertise of multiple stakeholders, including PhD career development professionals, employers of PhDs, leaders in graduate and postdoctoral education, funders, and policy makers. As more and more institutions join ranks in providing career and professional development for a wide range of career choices, it makes sense to accurately track and report on the outcomes of alumni along those career paths. It is our hope that the growing momentum on this front will provide enormous benefits to policy makers, employers, training programs, career development professionals, funders, and -- most of all -- current and future trainees.\n\n\nData availability\n\nIRB approval for public data-sharing is limited to de-identified and aggregated data only, due to concerns of sharing personally identifiable information, which could be traced back to identify individuals included in the data set. The personally-identifying job title, employer, and LinkedIn profile or other job-related URL were collected by individual institutions for their own alumni and used for coding purposes. Limited data-sharing for publication was approved by the respective IRBs as noted above, along with internal data-sharing agreements with NIH and the BEST Consortium.\n\nOpen Science Framework: Applying inter-rater reliability to improve consistency in classifying PhD career outcomes - Extended Data S1-S11, https://doi.org/10.17605/OSF.IO/5CGUJ35.\n\nThis project contains the following extended data:\n\nS1. Unified Career Outcomes Taxonomy Experimental Version 2 (UCOT Exp2) unabridged\n\nS2. Annotated table of changes made to variations of UCOT 2017 in generating UCOT Exp2\n\nS3. Unified Career Outcomes Taxonomy Experimental Version 2 (UCOT Exp2) Final Guidance Document and Crosswalk\n\nS4. Selection and Composition of Alumni Records for Experiment 1 Data Set\n\nS5. Selection and Composition of Alumni Records for Experiment 2 Data Set\n\nS6. Two measures of discordance used to compare coding after Experiments 1 and 2\n\nS7. Data Collection Workbook template.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nWhile it is not possible to share the data that includes the identifying information, de-identified coder-agreement raw data is available alongside the extended data files in the OSF project (https://doi.org/10.17605/OSF.IO/5CGUJ35):\n\nS8. De-identified coder agreement data for Experiment 1 Data Set.\n\nS9. Coding Key for Experiment 1.\n\nS10. De-identified coder agreement data for Experiment 2 Data Set.\n\nS11. Coding Key for Experiment 2\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank members of the BEST Consortium, especially Julia Melkers, Liz Silva, Bill Lindstaedt, and Spencer Fenn for their valuable feedback on experimental design, data interpretation, and comments on earlier versions of this manuscript. We are grateful to Ambika Mathur, Roger Chalkley, and Fred Meyers for their foundational work in this field and continued support of this project. We thank Patricia Labosky, Rebecca Lenzi, and members of the BEST External Scientific Panel (comprised of Holly Falk-Krzesinski, Diane Klotz, Kenneth I. Maynard, Jeri Janowsky, and Jay George) for their advocacy of this initiative. We thank the Odum Institute for Research in Social Science at the University of North Carolina at Chapel Hill for consultations on our statistical analyses, Brandon Barnes (Emory University) for his early contributions to data collection and curation methodology, and the Center for Postdoctoral Affairs in the Health Sciences at University of Pittsburgh for their thoughtful comments. The conclusions, views, and opinions expressed in this study are those of the authors and do not necessarily reflect the official policy or position of NIH, the institutions employing the authors, nor the BEST consortium as a whole.\n\nA previous version of this work is available from: https://doi.org/10.1101/370783.\n\n\nReferences\n\nNational Institutes of Health: Biomedical Research Workforce Working Group Report. Bethesda: National Institutes of Health. 2012. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKimble J, Bement WM, Chang Q, et al.: Strategies from UW-Madison for rescuing biomedical research in the US. eLife. 2015; 4: e09305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTracking Participation and Evaluating Outcomes. In: NIH BEST Consortium. Nashville: NIH Best Consortium; c2014-2018 [cited 12 Jul 2018]. Reference Source\n\nMathur A, Brandt P, Chalkley R, et al.: Evolution of a Functional Taxonomy of Career Pathways for Biomedical Trainees. J Clin Transl Sci. 2018; 2(2): 63–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlank R, Daniels RJ, Gilliland G, et al.: A new data effort to inform career choices in biomedicine. Science. 2017; 358(6369): 1388–1389. PubMed Abstract | Publisher Full Text\n\nUnderstanding PhD Career Pathways for Program Improvement. In: Council of Graduate Schools. Washington, DC. 2015 [cited 12 Jul 2018]. 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Reference Source\n\nMathur A, Cano A, Kohl M, et al.: Visualization of gender, race, citizenship and academic performance in association with career outcomes of 15-year biomedical doctoral alumni at a public research university. PLoS One. 2018; 13(5): e0197473. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu H, Gilliam RST, Peddada SD, et al.: Visualizing detailed postdoctoral employment trends using a new career outcome taxonomy. Nat Biotechnol. 2018; 36(2): 197. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilva EA, Des Jarlais C, Lindstaedt B, et al.: Tracking Career Outcomes for Postdoctoral Scholars: A Call to Action. PLoS Biol. 2016; 14(5): e1002458. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFuhrmann CN, Hobin JA, Lindstaedt B, et al.: myIDP, an online interactive career planning tool. AAAS (American Association for the Advancement of Science). Website launched Sept. 7, 2012. Reference Source\n\nStayart CA, Hutto T, Baas T, et al.: Constructing a Taxonomy for Career Outcome Reporting. In iCollaborative. 2017 [cited 12 Jul 2018]. Reference Source\n\nStayart CA, Mathur C, Chalkley R, et al.: Report from meeting on career outcomes. In: NIH BEST. 2017 [cited 12 Jul 2018]. Reference Source\n\nPickett C: Improving transparency in PhD career outcomes. In: Rescuing Biomedical Research. 2017 [Cited 12 July 2018]. Reference Source\n\nSilva EA, Meja AB, Watkins ES: Where do our graduates go? A toolkit for retrospective and ongoing career outcomes data collection for biomedical PhD students and postdoctoral scholars. bioRxiv. 2019. Publisher Full Text\n\nKrippendorff K: Content Analysis: An introduction to its methodology. 3rd ed. Everly Hills, CA: Sage. 2013. Reference Source\n\nHallgren KA: Computing Inter-Rater Reliability for Observational Data: An Overview and Tutorial. Tutor Quant Methods Psychol. 2012; 8(1): 23–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTaylor J, Watkinson D: Indexing reliability for condition survey data. The Conservator. 2007; 30(1): 49–62. Publisher Full Text\n\nHayes AF, Krippendorff K: Answering the call for a standard reliability measure for coding data. Communication Methods and Measures. 2007; 1(1): 77–89. Publisher Full Text\n\nCarnegie Classification of Institutions of Higher Education 2015 Update Public File. In: Indiana University Center for Postsecondary Research. Bloomington, IN. 2015 [cited 13 Jul 2018]. Reference Source\n\nNPA Responds to NAS’ The Postdoctoral Experience Revisited. 2014 [cited 12 Jul 2018]. In: National Postdoctoral Association. Reference Source\n\nPostdoc Policy Issues - A Brief Overview. In: National Postdoctoral Association. [cited 13 Jul 2018]. Reference Source\n\nGardner L: Want to Kill Tenure? Be Careful What You Wish For. In: Chronicle of Higher Education. 2018 [cited 12 Jul 2018]. Reference Source\n\nMathur A, Lean SF, Walker NV, et al.: Career outcomes for STEM, social and behavioral sciences and education doctoral alumni. Ageing Sci Ment Health Stud. 2018; 2(3): 1–33. Reference Source\n\nDamarin SK: The mathematically able as a marked category. Gender Educ. 2000; 12(1): 69–85. Publisher Full Text\n\nStayart AC, Monsalve G, Brandt PD, et al.: Applying inter-rater reliability to improve consistency in classifying PhD career outcomes - Extended Data S1-S11. 2020. http://www.doi.org/10.17605/OSF.IO/5CGUJ"
}
|
[
{
"id": "58480",
"date": "23 Jan 2020",
"name": "Christopher L. Pickett",
"expertise": [
"Reviewer Expertise Science policy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Applying inter-rater reliability to improve consistency in classifying PhD career outcomes” by Stayart et al. examines how consistent different raters are when making taxonomic determinations of Ph.D. career types. Starting with a taxonomy developed by several groups and universities in 2017, the authors test the degree of agreement among six different coders classifying several hundred records. They then identified areas of disagreement among the coders and made critical definitional changes to the taxonomy that improve the degree of agreement across coders.\n\nThe paper by Stayart et al. is an important contribution to the effort to encourage universities to be more transparent about the careers their Ph.D.s take after they graduate. Improving the career taxonomy and strengthening the definitions therein, along with the authors’ discussion of the shortcomings of these taxonomic approaches, will make it easier for more institutions to transparently provide this kind of information to their trainees.\nMajor issue:\nThe text in the Discussion under the “Description of changes made to UCOT 2017 to increase reliability” reads as results rather than discussion. The authors should strongly consider moving this entire section into the Results section to help readers understand the real differences between UCOT Exp 1 and UCOT Exp 2 as well as the beneficial changes the authors made to their final taxonomy.\nMinor issues:\n“…a major impeding factor in this effort to share outcomes data has been the multitude of career outcomes taxonomies…” is not quite correct. The impediment, in my view, is not the taxonomies per se, but the small, yet important, differences in how career outcomes are binned, leading to real differences in taxonomies and data presentation. The authors echo this point of view in the Discussion when they caution institutions not about making new taxonomies, but about being forthright with any changes they make to the common taxonomy. The authors should consider recasting this sentence.\n\nIn the third paragraph of the Introduction, it may be useful to the reader to include an example of a job title that could potentially fall into more than one category.\n\nA basic diagram of the three tiers of UCOT 2017 as Fig. 1 may help readers understand how the scheme works and provide a firmer basis for understanding the experiments that follow. The authors present the three tiers in three separate tables, but it is difficult to understand from these tables how the tiers function together.\n\nIt would be useful for the authors to provide information on the degree of agreement among the coders after the second experiment. After Exp1, the authors remark “The six coders in Experiment 1 had six-way agreement on 77% of all records within…” What percentage of records were coded the same by the coders in Exp2, and what were the areas of disagreement? Were the problems areas identified in Exp1 resolved in Exp2?\n\nThe authors should take a little more space to discuss the issue of time needed to code records. The authors report it takes coders about 1 min 40 seconds per record to code them; however, the average time for experienced and inexperienced coders comes out to be about a 30-second per record difference (328 min/219 records =~1.5 min; 429 min/219 records =~2 min). At this level of time, that’s an important difference!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5435",
"date": "28 Apr 2020",
"name": "Rebekah Layton",
"role": "Author Response",
"response": "The authors wish to thank our reviewers for their thoughtful comments and thorough review. Please find a point-by-point response below. R denotes reviewer comments, A denotes author responses. --- R2: Reviewer 2 - Overall Comments: “The article “Applying inter-rater reliability to improve consistency in classifying PhD career outcomes” by Stayart et al. examines how consistent different raters are when making taxonomic determinations of Ph.D. career types. Starting with a taxonomy developed by several groups and universities in 2017, the authors test the degree of agreement among six different coders classifying several hundred records. They then identified areas of disagreement among the coders and made critical definitional changes to the taxonomy that improve the degree of agreement across coders.” “The paper by Stayart et al. is an important contribution to the effort to encourage universities to be more transparent about the careers their Ph.D.s take after they graduate. Improving the career taxonomy and strengthening the definitions therein, along with the authors’ discussion of the shortcomings of these taxonomic approaches, will make it easier for more institutions to transparently provide this kind of information to their trainees.” A2: Authors response to Reviewer 2 - Overall Comments: We thank Reviewer 2 for noting the importance of developing a culture of transparency around career outcomes, to which we hope this paper contributes. The authors main purpose in executing the iterative experiment was, indeed, to ultimately serve the practitioner responsible for coding the information. We also believe that the benefit of more reliable and consistent classifications will trickle down to important stakeholders, including prospective and current trainees, who will consequently be empowered with high-quality data to confidently make their own informed career decisions. Furthermore, policy changes are often instigated by data – without robust, reproducible, and reliable data, it is difficult to advocate for evidence-based policy changes around career and professional development. R2.1. Major: “The text in the Discussion under the “Description of changes made to UCOT 2017 to increase reliability” reads as results rather than discussion. The authors should strongly consider moving this entire section into the Results section to help readers understand the real differences between UCOT Exp 1 and UCOT Exp 2 as well as the beneficial changes the authors made to their final taxonomy.” A2.1 Major: Thank you for this excellent suggestion, we agree that this would strengthen the flow and readability of the paper without sacrificing content. We have adopted this change as recommended and moved this section into the results. R2.2 Minor issues: “…a major impeding factor in this effort to share outcomes data has been the multitude of career outcomes taxonomies…” is not quite correct. The impediment, in my view, is not the taxonomies per se, but the small, yet important, differences in how career outcomes are binned, leading to real differences in taxonomies and data presentation. The authors echo this point of view in the Discussion when they caution institutions not about making new taxonomies, but about being forthright with any changes they make to the common taxonomy. The authors should consider recasting this sentence. A2.2: Thank you for sharing this helpful perspective. We have changed the wording to reflect this suggestion. We changed the original sentence from, “However, a major impeding factor in this effort to share outcomes data has been the multitude of career outcomes taxonomies developed by individual institutions and agencies.”- To: - “However, a factor that has impacted the sharing of outcomes data has been a confusion over which career outcomes taxonomy is being displayed in which context.” R2.3: “In the third paragraph of the Introduction, it may be useful to the reader to include an example of a job title that could potentially fall into more than one category.” A2.3: Thank you for pointing out the need for an example here, we agree that this would strengthen the text and have added the following accordingly: “For example, a Program Director title at a University could have very different job responsibilities compared to a person of the same title in a life science consulting firm.” R2.4 Minor issues: “A basic diagram of the three tiers of UCOT 2017 as Fig. 1 may help readers understand how the scheme works and provide a firmer basis for understanding the experiments that follow. The authors present the three tiers in three separate tables, but it is difficult to understand from these tables how the tiers function together.” A2.4: We agree that a figure with a broad level overview of how the tiers fit together would be helpful to the reader. In our attempt to do this in the past we found it difficult to maintain fonts that were readable in a small amount of space. Hence, we were unable to create a visual display that fit effectively on a manuscript page to illustrate this. Furthermore, in order to represent all variations, we would need multiple iterations of the figure, which may not be a good use of space (duplicative), or worse yet, could be confusing as to which variant is being discussed. Hence, after repeated failed attempts, we concluded that an effective visualization was not feasible. R2.5 Minor issues: “It would be useful for the authors to provide information on the degree of agreement among the coders after the second experiment. After Exp1, the authors remark “The six coders in Experiment 1 had six-way agreement on 77% of all records within…” What percentage of records were coded the same by the coders in Exp2, and what were the areas of disagreement? Were the problems areas identified in Exp1 resolved in Exp2? A2.5: We explored using ‘coder-agreement’ as a metric, however concluded that there were inherent limitations of this method and elected to use a more robust measure of coder agreement, Krippendorf’s alpha. Using this more sophisticated measured, we hoped to be able to compare it to acceptable levels of agreement as established and validated in previous literature (unrelated to taxonomies). Furthermore, use of Krippendorf’s alpha avoids inherent flaws in the simpler ‘coder-agreement’ metric, such as potentially overestimating agreement by not taking ‘agreement by chance’ into account. We believe that Krippendorf’s alpha permitted a more robust comparison across both studies. R2.6 Minor issues: “The authors should take a little more space to discuss the issue of time needed to code records. The authors report it takes coders about 1 min 40 seconds per record to code them; however, the average time for experienced and inexperienced coders comes out to be about a 30-second per record difference (328 min/219 records =~1.5 min; 429 min/219 records =~2 min). At this level of time, that’s an important difference!” A2.6: Based on the reviewer’s suggestion, we have addressed in greater detail what this would mean in real time for experienced versus inexperienced coders, and added the following example to the text: “The total time for experienced coders ranged from 213 to 540 minutes, on average 328 minutes, or about a minute and a half. In comparison, the total time for inexperienced coders ranged from 246 to 545 minutes coders, on average 429 minutes, or about two minutes. While this may not seem like a huge difference in time to code records for inexperienced versus experienced coders, depending on the size of one’s alumni populations, this may affect one’s approach. For instance, if binning 1000 records, taking the time to become a trained “experienced coder” would save each coder approximately a day’s worth of full time effort (e.g., approximately 33 hours versus 25 hours).”"
}
]
},
{
"id": "58478",
"date": "10 Feb 2020",
"name": "Reinhart Reithmeier",
"expertise": [
"Reviewer Expertise Graduate Professional Development",
"Teaching",
"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 paper describes the results of an important pilot study using 800 alumni records to develop a uniform taxonomy to classify career outcomes of PhD graduates across institutions. Success in developing such a survey instrument will allow uniform reporting, aggregation of data and meaningful comparisons. Attempts were made to address ambiguities in some categories of the taxonomy, including the addition of a flag system. There still remained discordance among the coders even among those who went through a training exercise. The inter-rater reliability did not improve significantly from Experiment 1 to 2. In career outcomes there is the constant challenge to balance the use of broad categories and the degree of granularity necessary to generate meaningful data.\n\nComments:\nIn Table 1 the Workforce Sectors are broad: Academia, Government, For-Profit, Non-Profit, Other. This classification is based mainly on the nature of the Employer and should be fairly straight-forward to bin. Consider adding Self-employed. Workforce Sector would be a Level 1 Bin.\n\nThe next level of classification within each Workforce Sector could be based on the Job Title, rather than Career Type and Job Function. This would be a Level 2 Bin and easy to code from drop-downs of typical job titles. A Job Title is easier to bin and often provides a good definition of the Job Function. A challenge that needs to be addressed are multiple job functions. In this regard Professor as a Job Title is an ideal career type as they all carry out three main job functions: teaching, research and administration all to various extents. Some Professors are also entrepreneurs and start companies.\n\nThe use of Career Type: Primarily Research or Primarily Teaching at this level is not useful. Indeed, over a career trajectory the mix of teaching, research and administration for a Professor may change, yet the Career Type and does not. Determining whether a professor is primarily Research or Primarily Teaching could be difficult to determine, even based on the Carnegie classification of Institutions. Similarly, in the Private Sector the Job Title: Research Scientist could describe a dynamic mix of research and administration typical of people working in this sector. Placing individuals in the proper Job Titles using pre-populated drop-down menus should be straight-forward.\n\nThe current Table 3 Job Function is a muddle of job titles and job functions. I would not put Deceased and Retired under the same function!\n\nThe Level 3 Bin could now be Job Function under Job Title. This could gleaned from a search of the LinkedIn profile for pre-determined key words. Here, job functions such as Research, Teaching or Administration could be useful. Using the term primarily may be difficult to assess, other than what is already obvious for the Job Title (e.g., Manager, Director, V-P, etc.).\n\nFurther Training or Education should include Post-doctoral Fellow and Professional Schools as the two main categories where PhDs go.\n\nThe average time to classify at under 2 minutes seems very short.\n\nThe goal of this project involves the classification of thousands upon thousands of PhD alumni (big data) from institutions across the USA and is an ideal application of AI through machine learning to find meaningful patterns and classifications into bins. It would be useful to compare the results of the current project with the results obtained by computer neural networks using the same training data set.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5436",
"date": "28 Apr 2020",
"name": "Rebekah Layton",
"role": "Author Response",
"response": "The authors wish to thank our reviewers for their thoughtful comments and thorough review. Please find a point-by-point response below. R denotes reviewer comments, A denotes author responses. --- R1: Reviewer 1 - Overall Comment: “This paper describes the results of an important pilot study using 800 alumni records to develop a uniform taxonomy to classify career outcomes of PhD graduates across institutions. Success in developing such a survey instrument will allow uniform reporting, aggregation of data and meaningful comparisons. Attempts were made to address ambiguities in some categories of the taxonomy, including the addition of a flag system. There still remained discordance among the coders even among those who went through a training exercise. The inter-rater reliability did not improve significantly from Experiment 1 to 2. In career outcomes there is the constant challenge to balance the use of broad categories and the degree of granularity necessary to generate meaningful data.” A1: Author Response to Reviewer 1 - Overall Comment: We thank the reviewer for a thoughtful and well-considered response. Although the gains in reliability were not dramatic, the reliability increased in every tier using the latter version (UCOT Exp2); more importantly, each of these tiers then met generally-accepted criteria for adequate reliability (at least with experienced coders). As noted, we agree that it is important to balance detail versus usability (granularity vs. breadth). We believe that UCOT Exp2 provides an example of how even slight modifications to wording and training can affect interpretation and reliability of the taxonomy, and that our experimental data demonstrate the necessity of further evolution of taxonomies as the workforce itself evolves. R1.1: “In Table 1 the Workforce Sectors are broad: Academia, Government, For-Profit, Non-Profit, Other. This classification is based mainly on the nature of the Employer and should be fairly straight-forward to bin. Consider adding Self-employed. Workforce Sector would be a Level 1 Bin.” A1.1: In response to the reviewer’s comment regarding Workforce Sector organization and inclusion of self-employment therein, we agree that self-employment is not accurately captured in UCOT Exp2 and that, since self-employment is becoming a more frequent career outcome for PhD-trained scientists, we must identify a way to represent it in the taxonomy. During our experiment, we did in fact consider the importance of self-employment, but realized that every form of self-employment could be classified within an existing sector of the taxonomy. For instance, the Workforce Sector a self-employed individual could be for-profit or non-profit (Tier 1), and, within that, their Job Type could be in any type (e.g., focused on teaching, research, or science-related; Tier 2), and finally, the nature of their self-employment would be appropriately captured in Job Function (Tier 3) as Entrepreneur. In our second iteration of the experiment (UCOT Exp2), we advised that self-employed individuals be classified in the field of their specialization (if freelance) or as entrepreneur (in Tier 3). However, that solution inadequately captures the important distinction of self-employment. Prompted by your comment, we have considered how self-employment could be introduced to the taxonomy while still satisfying taxonomic logic, since if a record can be classified within multiple categories in the same tier, an additional level of classification is required to capture the specialization of the record. We propose that, in Job Function, self-employed individuals continue to be classified by the field in which they are working (for example, communications), and that their self-employment characteristic could be captured by a flag system, similar to that used with faculty and postdoctoral scientists. We have now incorporated this as a recommendation in future directions. The following has been added to the text: “This iteration of the career taxonomy does not adequately capture the characteristic of self-employment, since self-employed individuals are currently classified by the field in which they are working. When the taxonomy was developed, the original UCOT designers agreed that when a classification could fall within multiple categories in the same tier, it should be considered subsequently in a different tier. In future variations, self-employment could be captured by an additional flag system similar to faculty or postdocs, which may be relevant to institutions and programs with an interest in entrepreneurship startups, and spinoffs. R1.2 & 1.5: “The next level of classification within each Workforce Sector could be based on the Job Title, rather than Career Type and Job Function. This would be a Level 2 Bin and easy to code from drop-downs of typical job titles. A Job Title is easier to bin and often provides a good definition of the Job Function. A challenge that needs to be addressed are multiple job functions. In this regard Professor as a Job Title is an ideal career type as they all carry out three main job functions: teaching, research and administration all to various extents. Some Professors are also entrepreneurs and start companies….” “…The Level 3 Bin could now be Job Function under Job Title. This could gleaned from a search of the LinkedIn profile for pre-determined key words. Here, job functions such as Research, Teaching or Administration could be useful. Using the term primarily may be difficult to assess, other than what is already obvious for the Job Title (e.g., Manager, Director, V-P, etc.).” A1.2& 1.5: Thank you for your thoughtful consideration of the structure of the taxonomy. We fully agree that starting with job titles seems straight forward and, historically, many career taxonomies started from that point, including our own. However, when applied to thousands of records by multiple individuals, we found an absence of convergence upon “typical” job titles due to the rapidly diversifying career pathways pursued by PhD-trained scientists. Indeed, it was the proliferation of job titles and their selective redefinition among industries that led to our observation that initial classification by job title actually contributed to inconsistent classification. This resulted in our proposal of an experiment in which we tested how well multiple independent coders could bin the same job title; we found that overreliance on job title was associated with unreliable coding (see V1, P13 Description of Changes to UCOT2017 for further discussion). R1.3: The use of Career Type: Primarily Research or Primarily Teaching at this level is not useful. Indeed, over a career trajectory the mix of teaching, research and administration for a Professor may change, yet the Career Type and does not. Determining whether a professor is primarily Research or Primarily Teaching could be difficult to determine, even based on the Carnegie classification of Institutions. Similarly, in the Private Sector the Job Title: Research Scientist could describe a dynamic mix of research and administration typical of people working in this sector. Placing individuals in the proper Job Titles using pre-populated drop-down menus should be straight-forward. A1.3: Our key reason for classifying career type as research/teaching/science-related is the important implications for the types of career and professional development activities that could be pursued by trainees during the training period in order to be best prepared for that type of career. For example, PhD students may not need to pursue postdoctoral training if their intended career does not directly involve research, or those who are interested in primarily-teaching careers should pursue additional training in educational pedagogy and classroom teaching. Similarly, we have observed a tendency among faculty and trainees alike to assume that a professorial job title implies that the individual bears the research responsibilities of a Principal Investigator. By relying on job titles alone as a primary classification strategy, we sacrifice valuable detail regarding the actual function of faculty positions across the types of universities and colleges. We feel that failing to differentiate ‘primarily research’ from ‘primarily teaching’ does a disservice to the career development of trainees, and thus it is important to capture the primary work activity of the individual. Finally, many institutions and funding agencies place value in distinguishing between career outcomes that could be classified as ‘research-focused’ roles in higher education versus those that are ‘teaching-focused.’ Hence, we believe that this distinction is worth including and aids in interpretation and display of the data. R1.4: The current Table 3 Job Function is a muddle of job titles and job functions. I would not put Deceased and Retired under the same function! A1.4: We recognize the often-crucial role that experienced retirees can play in organizations, whether formal or informal. However, with regards to the functional use of the career taxonomy, the commonality between retirees and deceased individuals is the removal of their formal full-time effort from the economy. We group them together to distinguish them from individuals who are “Unemployed or Seeking Employment,” because the latter contribute to the unemployment rate for scientists. Nonetheless, if someone were interested in additional granularity, more detail could be tracked in institutional databases to distinguish between the two. R1.6: “Further Training or Education should include Post-doctoral Fellow and Professional Schools as the two main categories where PhDs go.” A1.6: We struggled with the decision to separate postdoctoral positions from ‘further training or education’ because a postdoctoral period is one during which the individual is obtaining further training in a specific career. However, upon reflection, we concluded that a postdoctoral training period is actually a blended role of trainee and employee, which primarily consists of learning on-the-job rather than formal coursework. Hence, in our view, postdoctoral fellowship shares more in common with employment than education. While postdoctoral training is also a learning period, we make a further distinction in our definitions that inclusion in “Further Education and Training” connotes the completion of coursework towards a formal degree or certification. Furthermore, by adopting postdoctoral trainees as a Job Function (acknowledging that it is with the dual role of training), we are able to maintain an important level of granularity to the ever-neglected category of postdoctoral trainee, whose roles often remain unexamined due to their complexity. In accordance with the reporting requirements of some national organizations (e.g., CNGLS), currently many institutions choose only to report Tiers 1 & 2. Given the probability of use of the taxonomy without including Tier 3- and given the increased availability of non-academic and non-research postdoctoral positions in areas such as policy, communications, teaching, industry, and more – we felt the visibility of non-traditional training roles would otherwise be compromised, leaving them for all intents and purposes, invisible. Due to the formal “Further Training or Education” definition adopted, and the need to capture different types of postdoctoral training, we remain committed to this structural change from previous versions, despite the controversial nature of this recommendation (see p12, “Description of Changes Made to Increase UCOT Reliability” for additional discussion). Regardless of any group’s decision for classifying postdoctoral fellows, we strongly advise taxonomy users to provide transparency and consistency of definition within appropriate context (e.g., if reporting for CNGLS, clearly label the version being reported). This may at times result in multiple sets of reports depending upon the number of taxonomic tiers an institution chooses to report, however we hope that over time, as institutions build baseline career outcomes data, it will become easier to update existing data sets with greater levels of detail if greater granularity if so desired. R1.7: “The average time to classify at under 2 minutes seems very short.” A1.7: We were also pleased with the very reasonable time per record, as averaged over multiple coders who each tracked time spent per number of records to provide this information (for further discussion see V1, P10, Results Section). We would like to clarify that this time reflects only the actual classification of the record, not the time required to find the individual. We also underline that all coders used in our study were PhD-trained program directors in graduate education, which imbues a bias of familiarity with career outcomes, job titles, and job functions that are typical among PhD-trained scientists. In future directions, we suggest that comparisons of time and accuracy produced by other types of coders should be explored, and that, in all cases, teams should be trained as a unit to achieve maximum concordance within the unit. R1.8: “The goal of this project involves the classification of thousands upon thousands of PhD alumni (big data) from institutions across the USA and is an ideal application of AI through machine learning to find meaningful patterns and classifications into bins. It would be useful to compare the results of the current project with the results obtained by computer neural networks using the same training data set.” A1.8: The authors concur that this would be an interesting iteration for future studies to explore. As insights from big data have transformed much of our current economy and society, there are most certainly interesting applications for use of machine learning and big data to apply to PhD career outcomes. With that said, there are limitations to technological applications in their ability to distinguish nuances between these categories or consider situational differences. Future experiments could use machine learning to develop a taxonomy de novo, based in its own determination of which parameters, definitions, searches, etc. would create the taxonomic tiers. Indeed, applying a neural networking model to generate a career taxonomy would be fascinating! In the interim, comparisons of human vs. machine performance, or hybrid human/computer coding might be a good initial exploration. In response to the reviewer’s comment, we have introduced the following text to our manuscript: “While this experiment focused on evaluating the consistency and time investment needed for manually classifying career outcomes, future directions to improve efficiency might include development of automated systems or programs to implement career classifications, particularly with the advent of machine learning in the current era. As a first step, perhaps machine-learning could increase efficiency by creating initially proposed binning of career outcomes which could then be spot-checked or confirmed by a human being on a second pass. Perhaps this process could even be fully automated pending technological advances.”"
}
]
}
] | 1
|
https://f1000research.com/articles/9-8
|
https://f1000research.com/articles/9-113/v1
|
13 Feb 20
|
{
"type": "Research Article",
"title": "Machine learning meets pKa",
"authors": [
"Marcel Baltruschat",
"Paul Czodrowski",
"Marcel Baltruschat"
],
"abstract": "We present a small molecule pKa prediction tool entirely written in Python. It predicts the macroscopic pKa value and is trained on a literature compilation of monoprotic compounds. Different machine learning models were tested and random forest performed best given a five-fold cross-validation (mean absolute error=0.682, root mean squared error=1.032, correlation coefficient r2 =0.82). We test our model on two external validation sets, where our model performs comparable to Marvin and is better than a recently published open source model. Our Python tool and all data is freely available at https://github.com/czodrowskilab/Machine-learning-meets-pKa.",
"keywords": [
"machine learning",
"pKa value",
"protonation",
"dissociation"
],
"content": "Introduction\n\nThe acid-base dissociation constant (pKa) of a drug has a far-reaching influence on pharmacokinetics by altering the solubility, membrane permeability and protein binding affinity of the drug. Several publications summarize these findings in a very comprehensive manner1–7. An accurate estimation of pKa values is therefore of utmost importance for successful drug design. Several (commercial and non-commercial) tools and approaches for small molecule pKa prediction are available: MoKa8 uses molecular interaction fields, whereas ACD/Labs Percepta Classic9, Marvin10 and Epik11 make use of the Hammet-Taft equation. By means of Jaguar12, a quantum-mechanical approach to pKa prediction becomes possible. Recently, the usage of neural nets for pKa prediction became prominent13–15. In particular, the publication by Williams et al.15 make use of a publicly available data set provided by the application DataWarrior16 and provides a freely available pKa prediction tool called OPERA.\n\nAs this article is part of a Python collection issue, we provide a pKa prediction method entirely written in Python17 and make it available open source (including all data). Our tool computes the macroscopic pKa value for a monoprotic compound. Our model solely differentiates between a base and acid based on the predicted pKa value; i.e., we do not offer separate models for acids and bases. In addition to pKa data from DataWarrior16, we also employ pKa data from ChEMBL18. As external validation sets, we use compound data provided by Novartis19 and a manually curated data set compiled from literature20–24, which are not part of the training data.\n\n\nMethods\n\nOne crucial point in the field of pKa measurements (and its usage for pKa predictions) is linked to the different experimental methods25,26. Based on the Novartis set, the correlation between capillary electrophoresis and potentiometric measurements (for 15 data points) is convincing enough (mean absolute error (MAE)=0.202, root mean squared error (RMSE)=0.264, correlation coefficient r2=0.981) for us to combine pKa measurements from these different experimental methods (see Figure 1).\n\nMAE, mean absolute error; RMSE, root mean square error.\n\nWe also compare the overlap of the filtered (see next section) ChEMBL and DataWarrior data sets, 187 monoprotic molecules could be identified in both sources. Due to the missing annotation, it remains unclear if different experimental methods were used or multiple measurements with the same experimental method have been performed (or a mixture of both). Either way, this comparison is an additional proof-of-concept that the ChEMBL and DataWarrior pKa data sources can be combined after careful curation. The aforementioned intersection is given in Figure 2. The correlation coefficient between the annotated pKa values for these two data sets r2 is 0.949, the MAE is 0.275, and the RMSE is 0.576.\n\nMAE, mean absolute error; RMSE, root mean square error.\n\nA ChEMBL18 web search was performed to find all assays containing pKa measurement data. The following restrictions were made: it must be a physicochemical assay, the measurements must be taken from scientific literature, the assay must be in \"small-molecule physicochemical format\" and the organism taxonomy must be set to \"N/A\". This results in a list of 1140 ChEMBL assays downloaded as CSV file. Using a Python script, the CSV file was read in and processed further, extracting all additional information required from an internally hosted copy of the ChEMBL database via SQL. Only pKa measurements, i.e. ChEMBL activities, were taken into account that were specified as exact (\"standard_relation\" equals \"=\") and for which one of the following names was specified as \"standard_type\": \"pka\", \"pka value\", \"pka1\", \"pka2\", \"pka3\" or \"pka4\" (case-insensitive). Measured values for which the molecular structure was not available were also sorted out. The resulting 8111 pKa measured values were saved as SDF file.\n\nA flat file from DataWarrior16 named “pKaInWater.dwar” was used in addition. In this case, the file was converted to an SDF file only and contains 7911 entries with valid molecular structures.\n\nThese data sets were concatenated for the purpose of this study and preprocessed as follows:\n\nRemove all salts from molecules\n\nRemove molecules containing nitro groups, Bor, Selenium or Silicium\n\nFilter by Lipinski‘s rule of five (one violation allowed)\n\nKeep only pKa data points between 2 and 12\n\nTautomer standardization of all molecules\n\nProtonate all molecules at pH 7.4\n\nKeep only monoprotic molecules regarding the specified pKa range\n\nCombine data points from duplicated structures while removing outliers\n\nAll steps up to filtering out all pKa values outside the range of 2 to 12 were performed with Python and RDKit27. The QuacPac28 Tautomers tool from OpenEye was used for tautomer standardization and setting the protonation state to pH 7.4. The Marvin10 tool from ChemAxon was used to filter out the multiprotic compounds. It predicted the pKa values of all molecules in the range 2 to 12 and then retained only those molecules where Marvin did not predict more than one pKa in that range.\n\nThe removal of the outliers is performed in two steps. First, before combining multiple measurements for the same molecules, all entries where the pKa predicted by ChemAxon's Tool Marvin differs from the experimental value by more than four log units are removed. All molecules were then combined on the basis of the isomeric SMILES. In the second step, when combining several measured values of a molecule, all those values that deviate from the mean value by more than two standard deviations are removed. The remaining values are arithmetically averaged.\n\nAfter all, 5994 unique monoprotic molecules with experimental pKa values remained. The distribution of pKa values is given in Figure 3. The same preprocessing steps were also performed on an external test data set provided to us by Novartis19 (280 molecules) and a manual curation (123 molecules) from literature20–24.\n\nFirst, to simplify cross-validation, a class \"CVRegressor\" was defined, which can serve as a wrapper for any regressor implementing the Scikit-Learn29 interface. This class simplifies cross-validation itself, training and prediction with the cross-validated model. Next, 196 of the 200 available RDKit descriptors (\"MaxPartialCharge\", \"MinPartialCharge\", \"MaxAbsPartialCharge\" and \"MinAbsPartialCharge\" were not used because they are computed as \"NaN\" for many molecules), and a 4096-bit long MorganFeature fingerprint with radius 3 were calculated for the training data set. Random forest (RF), support vector regression (SVR, two configurations), multilayer perceptron (MLP, three configurations) and XGradientBoost (XGB) were used as basic regressors. Unless otherwise specified, the Scikit-Learn default parameters (version 0.22.1) were used. For the RF model, only the number of trees was increased to 1000. For SVR the size of the cache was increased to 4096 megabytes in the first configuration, but this only increases the training speed and has no influence on the model quality. In the second configuration the parameter \"gamma\" was additionally set to the value \"auto\". For MLP in the first configuration the number of hidden layers was increased to two and the number of neurons per layer to 500. In the second configuration, early stopping was additionally activated, where 10% of the training data is separated as validation data. If the error of the validation data does not improve by more than 0.001 over ten training epochs, the training is stopped early to avoid overtraining. In the third configuration three hidden layers with a size of 250 neurons each were used with early stopping still activated. For XGB the default parameters of the used library XGBoost (version 0.90)30 were applied. The training of RF, MLP and XGB was parallelized on 12 CPU cores and the generation of the folds for cross-validation as well as the training itself were random seeded to a value of 24 to ensure reproducibility. This results in a total of seven different machine learning configurations.\n\nSix different descriptor/fingerprint combinations were also tested. First only the RDKit descriptors, followed by only the fingerprints and finally both combined. Additionally, all three combinations were tested again in a standardized form (z-transformed). As a result, 42 combinations of regressor and training data configurations were compared.\n\nA 5-fold cross-validation was performed for all configurations, which were evaluated using the MAE, RMSE and the empirical coefficient of determination (r2). After training was completed for all configurations, two external test data sets, which do not contain training data, were used to re-validate each trained cross-validated model. Here, MAE, RMSE, and r2 were also calculated as statistical quality measures.\n\nThe Python dependencies are as follows: Python >= 3.7, NumPy >= 1.18, Scikit-Learn >= 0.22, RDKit >= 2019.09.3, Pandas >= 0.25, XGBoost >= 0.90, JupyterLab >= 1.2, Matplotlib >= 3.1, Seaborn >= 0.9\n\nFor the data preparation pipeline, ChemAxon Marvin10 and OpenEye QUACPAC/Tautomers28 are required. To use the provided prediction model with the included Python script, ChemAxon Marvin10 is not required.\n\nFirst of all you need a working Miniconda/Anaconda installation. You can get Miniconda at https://conda.io/en/latest/miniconda.html.\n\nNow you can create an environment named \"ml_pka\" with all needed dependencies and activate it with:\n\n\n\nYou can also create a new environment by yourself and install all dependencies without the environment.yml file:\n\n\n\nIn case of Linux or macOS:\n\n\n\nIn case of Windows:\n\n\n\nPrediction pipeline. To use the data preparation pipeline you have to be in the repository folder and your conda environment have to be activated. Additionally the Marvin10 commandline tool cxcalc and the QUACPAC28 commandline tool tautomers have to be set in your PATH variable.\n\nAlso the environment variables OE_LICENSE (containing the path to your OpenEye license file) and JAVA_HOME (referring to the Java installation folder, which is needed for cxcalc) have to be set.\n\nAfter preparation you can display a small usage information with bash run_pipeline.sh -h. Examplary call:\n\n\n\nPrediction tool. First of all you have to be in the repository folder and your conda environment have to be activated. To use the prediction tool you have to retrain the machine learning model. Therefore just call the training script, it will train the 5-fold cross-validated Random Forest machine learning model using 12 cpu cores. If you want to adjust the number of cores you can edit the train_model.py by changing the value of the variable EST_JOBS.\n\n\n\nTo use the prediction tool with the trained model QUACPAC/Tautomers have to be available as it was mentioned in the section above.\n\nNow you can call the python script with an SDF file and an output path:\n\n\n\nIt should be noted that this model was built for monoprotic structures regarding a pH range of 2 to 12. If the model is used with multiprotic structures, the predicted values will probably not be correct.\n\n\nResults\n\nThe statistics for a five-fold cross-validation are given in Table 1. In terms of the mean absolute error, a random forest with scaled MorganFeatures (radius=3) and descriptors gives the best performing model (MAE=0.682, RMSE=1.032, r2=0.82). For the two external test sets (see Table 2), a random forest with FeatureMorgan (radius=3) gives the best model (Novartis: MAE=1.147, RMSE=1.513, r2=0.569; LiteratureCompilation: MAE=0.532, RMSE=0.785, r2=0.889). The predictive performance for Marvin10 and the OPERA tool15 are as follows: Novartis (Marvin: MAE=0.856, RMSE=1.166, r2=0.744; OPERA: MAE= 2.274, RMSE= 3.059, r2= -0.754) and LiteratureComplation20–24 (Marvin: MAE= 0.566, RMSE= 0.865, r2= 0.866; OPERA: MAE= 1.737, RMSE= 2.182, r2= 0.124). This shows that our model slightly outcompetes Marvin for the LiteratureCompilation, but Marvin performs better for the Novartis dataset. For both data sets, our models17 have a better predictive performance than the OPERA tool.\n\nThe two best and worst performing models are highlighted in green and red. For those neural networks where the values were specified as \"not available\" (“#NA”), the weights could not be optimized properly due to the large value range of the RDKit descriptors, so training failed here.\n\nMAE, mean absolute error; RMSE, root mean square error.\n\nThe two best and worst performing models are highlighted in green and red. For those neural networks where the values were specified as \"not available\" (“#NA”), the weights could not be optimized properly due to the large value range of the RDKit descriptors, so training failed here.\n\n*For OPERA 6 molecules from AvLiLuMoVe and 31 molecules from Novartis were left out because OPERA predicted either two or zero pKa values.\n\nMAE, mean absolute error; RMSE, root mean square error.\n\n\nDiscussion and conclusions\n\nThe good performance of Marvin on the Novartis set is interesting to note: the RMSE is almost 0.4 units better than our top performing model. This could be because Marvin’s training set is much larger than our own training set. This provides a better foundation for the training of the Marvin model. In contrast, Marvin performs slightly worse than our top model on the LiteratureCompilation. The OPERA tool performs significantly worse than our model on both external test sets. We assume that the addition of 2470 ChEMBL pKa-datapoints to our training set which are not part of the OPERA training set leads to this drop in predictive performance. In addition, the pre-processing of the data is performed differently by OPERA in comparison to our pre-processing procedure.\n\nAs next step for the enhancement and improvement of our pKa prediction model17, we are currently expanding it to multiprotic molecules. We are also investigating the impact of different neural net architectures and types (such as graph neural nets) and the development of individual models for acids and bases. From a chemistry perspective, an analysis of pKa effects of different functional groups (e.g. by means of matched molecular pairs analysis) is an on-going effort for a future publication.\n\n\nData availability\n\nZenodo: czodrowskilab/Machine-learning-meets-pKa article. https://doi.org/10.5281/zenodo.366224517.\n\nThe following data sets were used in this study:\n\nAvLiLuMoVe.sdf - Manually combined literature pKa data.\n\nchembl25.sdf - Experimental pKa data extracted from ChEMBL25.\n\ndatawarrior.sdf - pKa data shipped with DataWarrior.\n\ncombined_training_datasets_unique.sdf - Preprocessed and combined data from datasets chembl25.sdf and datawarrior.sdf, used as training dataset.\n\nAvLiLuMoVe_cleaned_mono_unique_notraindata.sdf - used as external testset.\n\nnovartis_cleaned_mono_unique_notraindata.sdf - inhouse dataset provided by Novartis19, used as external testset.\n\nThe data sets are also available at https://github.com/czodrowskilab/Machine-learning-meets-pKa.\n\nLicense: MIT license.\n\n\nSoftware availability\n\nThe source code is available at https://github.com/czodrowskilab/Machine-learning-meets-pKa.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.366224517.\n\nLicense: MIT license.",
"appendix": "Acknowledgments\n\nEd Griffen (MedChemica) is acknowledged for his investigations on our initial data set which revealed some wrongly annotated data points. We thank Bob Clark, Eric Jamois and Michael Lawless (Simulations Plus) for their support and advise in terms of data curation. Alpha Lee and Matt Robinson (Cambridge University) are appreciated for fruitful discussions.\n\n\nReferences\n\nManallack DT: The PKa Distribution of Drugs: Application to Drug Discovery. Perspect Medicin Chem. 2007; 1: 25–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManallack DT, Yuriev E, Chalmers DK: The influence and manipulation of acid/base properties in drug discovery. Drug Discov Today Technol. 2018; 27: 41–47. PubMed Abstract | Publisher Full Text\n\nManallack DT, Prankerd RJ, Yuriev E, et al.: The significance of acid/base properties in drug discovery. Chem Soc Rev. 2013; 42(2): 485–496. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeanwell NA: Improving drug candidates by design: a focus on physicochemical properties as a means of improving compound disposition and safety. Chem Res Toxicol. 2011; 24(9): 1420–1456. PubMed Abstract | Publisher Full Text\n\nGleeson MP: Generation of a set of simple, interpretable ADMET rules of thumb. J Med Chem. 2008; 51(4): 817–834. PubMed Abstract | Publisher Full Text\n\nLeeson PD, St-Gallay SA, Wenlock MC: Impact of Ion Class and Time on Oral Drug Molecular Properties. Med Chem Commun. 2011; 2(2): 91–105. Publisher Full Text\n\nCharifson PS, Walters WP: Acidic and basic drugs in medicinal chemistry: a perspective. J Med Chem. 2014; 57(23): 9701–9717. PubMed Abstract | Publisher Full Text\n\nMilletti F, Storchi L, Sforna G, et al.: New and original pKa prediction method using grid molecular interaction fields. J Chem Inf Model. 2007; 47(6): 2172–2181. PubMed Abstract | Publisher Full Text\n\nACD/Percepta, Advanced Chemistry Development, Inc.. Toronto, On Canada, Www.Acdlabs.Com, 2019. Reference Source\n\nMarvin 20.1.0, 2020. ChemAxon. Reference Source\n\nShelley JC, Cholleti A, Frye LL, et al.: Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des. 2007; 21(12): 681–691. PubMed Abstract | Publisher Full Text\n\nBochevarov AD, Watson MA, Greenwood JR, et al.: Multiconformation, Density Functional Theory-Based pKa Prediction in Application to Large, Flexible Organic Molecules with Diverse Functional Groups. J Chem Theory Comput. 2016; 12(12): 6001–6019. PubMed Abstract | Publisher Full Text\n\nFraczkiewicz R, Lobell M, Göller AH, et al.: Best of both worlds: combining pharma data and state of the art modeling technology to improve in Silico pKa prediction. J Chem Inf Model. 2015; 55(2): 389–397. PubMed Abstract | Publisher Full Text\n\nRoszak R, Beker W, Molga K, et al.: Rapid and Accurate Prediction of pKa Values of C-H Acids Using Graph Convolutional Neural Networks. J Am Chem Soc. 2019; 141(43): 17142–17149. PubMed Abstract | Publisher Full Text\n\nMansouri K, Cariello NF, Korotcov A, et al.: Open-Source QSAR Models for PKa Prediction Using Multiple Machine Learning Approaches. J Cheminform. 2019; 11(1): 60. Publisher Full Text | Free Full Text\n\nSander T, Freyss J, von Korff M, et al.: DataWarrior: an open-source program for chemistry aware data visualization and analysis. J Chem Inf Model. 2015; 55(2): 460–473. PubMed Abstract | Publisher Full Text\n\nLewis RA, Rodde S: Novartis Pharma AG. Basel, Switzerland.\n\nSettimo L, Bellman K, Knegtel RM: Comparison of the accuracy of experimental and predicted pKa values of basic and acidic compounds. Pharm Res. 2014; 31(4): 1082–1095. PubMed Abstract | Publisher Full Text\n\nLiao C, Nicklaus MC: Comparison of nine programs predicting pKa values of pharmaceutical substances. J Chem Inf Model. 2009; 49(12): 2801–2812. PubMed Abstract | Publisher Full Text\n\nAvdeef A: Absorption and Drug Development: Solubility, Permeability, and Charge State. John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2012. Publisher Full Text\n\nMorgenthaler M, Schweizer E, Hoffmann-Röder A, et al.: Predicting and tuning physicochemical properties in lead optimization: amine basicities. ChemMedChem. 2007; 2(8): 1100–1115. PubMed Abstract | Publisher Full Text\n\nLuan F, Ma W, Zhang H, et al.: Prediction of pKa for neutral and basic drugs based on radial basis function Neural networks and the heuristic method. Pharm Res. 2005; 22(9): 1454–1460. PubMed Abstract | Publisher Full Text\n\nDardonville C: Automated techniques in pKa determination: low medium and high-throughput screening methods. Drug Discov Today Technol. 2018; 27: 49–58. PubMed Abstract | Publisher Full Text\n\nReijenga J, van Hoof A, van Loon A, et al.: Development of Methods for the Determination of pKa Values. Anal Chem Insights. 2013; 8(1): 53–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRDKit, Open-Source Chemiformatics. Reference Source\n\nQUACPAC 2.0.2.2: OpenEye Scientific Software, Santa Fe, NM. Reference Source\n\nPedregosa F, Weiss R, Brucher M: Scikit-Learn: Machine Learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nChen T, Guestrin C: XGBoost: A Scalable Tree Boosting System. In Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining - KDD ’ 16; ACM Press: New York, New York, USA. 2016; 785–794. Publisher Full Text\n\nBaltruschat M, czodrowskilab, Czodrowski P: czodrowskilab/Machine-learning-meets-pKa article (Version article). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3662245\n\nGaulton A, Hersey A, Nowotka M, et al.: The ChEMBL database in 2017. Nucleic Acids Res. 2017; 45(D1): D945–D954. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "60011",
"date": "25 Feb 2020",
"name": "Ruth Brenk",
"expertise": [
"Reviewer Expertise chemoinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a machine learning method for pkA prediction. The presented method and results are scientifically solid. Therefore, I recommend the article for indexing. However, the methods and results should be presented more clearly before the article is indexed.\nThe following points should be addressed:\nPast tense should be used consistently to describe the methods and results.\n\nThe “different experimental methods” should be moved to the results part as it contains results.\n\nA short description of the molecules in the self-compiled data set and the Novartis set should be added. What type of molecules are contained in these sets?\n\nWhy does the user need to train the model before making predictions? Could the trained model be provided to make the developed tool easier to use?\n\nIn the discussion section, the developed method should be first discussed before discussing its performance relative to other methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5281",
"date": "27 Apr 2020",
"name": "Paul Czodrowski",
"role": "Author Response",
"response": "Past tense should be used consistently to describe the methods and results.We have used past tense throughout the entire document. The “different experimental methods” should be moved to the results part as it contains results.This part was moved accordingly. A short description of the molecules in the self-compiled data set and the Novartis set should be added. What type of molecules are contained in these sets?We have added this information in the manuscript: \"The Novartis data set consists of 280 unique molecules with a molecular weight between 129 and 670 daltons (mean value 348.68, standard deviation 94.17). The calculated LogP values vary between -1.54 and 6.30 (mean value 3.01, standard deviation 1.41). The 280 molecules spread over 228 unique Murcko Scaffolds. The ten most common murcko scaffolds cover 15% of the molecules of the total data set (42/280).\" Why does the user need to train the model before making predictions? Could the trained model be provided to make the developed tool easier to use?The user has to train the model again by himself or herself, because the model is larger than 1.7 GB as a file and therefore cannot be offered for download without paid services. Even with the best possible bzip2 compression, the file is still over 250 MB in size and thus larger than the 100 MB limit at GitHub. In addition, if the model is provided as a file, you must use exactly the same versions of the Python libraries used for training to load the model, otherwise inconsistencies may occur. However, if the model is trained again by yourself, only the minimum library requirements have to be met. In the discussion section, the developed method should be first discussed before discussing its performance relative to other methods.This was done and is reflected in the new manuscript version."
}
]
},
{
"id": "61511",
"date": "24 Mar 2020",
"name": "Johannes Kirchmair",
"expertise": [
"Reviewer Expertise cheminformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBaltruschat and Czodrowski report on the development of a Python-based tool for the prediction of pka values. The tool is made available open source and will certainly be useful to the scientific community.\nCould the authors please comment on the structural relationship between the training and the test data (how far apart are these datasets, e.g. measured by the distribution of pairwise Tanimoto coefficients based on molecular fingerprints)?\n\nCould the authors please confirm statistical significance of the key statements made in the first paragraph of the Results section, starting with \"This shows that our model slightly outcompetes...\"?\n\nIn the Abstract, please name the open source model that was tested as part of the comparative performance analysis.\n\nIt is not clear from the manuscript text whether capillary electrophoresis and potentiometric measurements were the only two experimental methods considered in this work (or, for the Novartis dataset only).\n\nPlease revise the sentence starting with \"We also compare the overlap...\".\n\n\"make use of a\" should be replaced by \"makes use of a\".\n\nPlease explain where DataWarrior's data originates from.\n\nPlease comment on the outliers observed in Figure 2.\n\nPlease explain the procedure that was followed to ensure that there was no overlap between the training data and the external validation sets (the Novartis dataset in particular).\n\n\"isomeric SMILES\": is it canonical as well?\n\nAvoid using \"you\" in the Implementation section (use \"one [can]...\" instead).\n\nThe part starting with \"For the two external test sets...\" is difficult to read. Could the authors find a more simple way to describe this and enable readers to compare the individual values 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?\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": "5429",
"date": "27 Apr 2020",
"name": "Paul Czodrowski",
"role": "Author Response",
"response": "Could the authors please comment on the structural relationship between the training and the test data (how far apart are these datasets, e.g. measured by the distribution of pairwise Tanimoto coefficients based on molecular fingerprints)?We have included new figures (2A and 2B) with pairwise Tanimoto coefficients (4096 bit MorganFeatures radius=3). Could the authors please confirm statistical significance of the key statements made in the first paragraph of the Results section, starting with \"This shows that our model slightly outcompetes...\"?A thorough test of the statistical significance is hindered by the fact the external open source tool OPERA cannot process all molecules of our external test sets. We therefore decided to rephrase “This showed that our model slightly outcompeted Marvin for the LiteratureCompilation” to “his showed that our model has a slightly better performance than Marvin for the LiteratureCompilation”. In the Abstract, please name the open source model that was tested as part of the comparative performance analysis.The open source model is now mentioned in the abstract. It is not clear from the manuscript text whether capillary electrophoresis and potentiometric measurements were the only two experimental methods considered in this work (or, for the Novartis dataset only).We mention this already in the section “Different experimental methods” :“Due to the missing annotation, it remained unclear if different experimental methods were used or multiple measurements with the same experimental method have been performed (or a mixture of both)”. Please revise the sentence starting with \"We also compare the overlap...\".Changed \"We also compared the overlap of the filtered (see next section) ChEMBL and DataWarrior data sets, 187 monoprotic molecules could be identified in both sources.\" To \"We also compared the pKa values of 187 monoprotic molecules contained in both the ChEMBL and DataWarrior data sets.\" \"make use of a\" should be replaced by \"makes use of a\".Done Please explain where DataWarrior's data originates from.We cannot make any statement about the origin, because it is not included in the DataWarrior file. Please comment on the outliers observed in Figure 2.We include the name of the outliers and the different pKa values originating from different data sources, if they differ by more than two log units.Please explain the procedure that was followed to ensure that there was no overlap between the training data and the external validation sets (the Novartis dataset in particular).To ensure that no training data was contained in the test data sets, the canonical isomeric SMILES were checked for matches in both training and test data sets and corresponding hits were removed from the test data sets. Chirality was not included in this comparison. \"isomeric SMILES\": is it canonical as well?Yes, it is the canonical isomeric SMILES generated by RDKit. Avoid using \"you\" in the Implementation section (use \"one [can]...\" instead).Done The part starting with \"For the two external test sets...\" is difficult to read. Could the authors find a more simple way to describe this and enable readers to compare the individual values more easily?We have pulled most of the numbers apart and included bullet points which improves the readability and makes a comparison more feasible."
}
]
}
] | 1
|
https://f1000research.com/articles/9-113
|
https://f1000research.com/articles/9-291/v1
|
24 Apr 20
|
{
"type": "Software Tool Article",
"title": "GoodVibes: automated thermochemistry for heterogeneous computational chemistry data",
"authors": [
"Guilian Luchini",
"Juan V. Alegre-Requena",
"Ignacio Funes-Ardoiz",
"Robert S. Paton",
"Guilian Luchini",
"Juan V. Alegre-Requena",
"Ignacio Funes-Ardoiz"
],
"abstract": "GoodVibes is an open-source Python toolkit for processing the results of quantum chemical calculations. Thermochemical data are not simply parsed, but evaluated by evaluation of translational, rotational, vibrational and electronic partition functions. Changes in concentration, pressure, and temperature can be applied, and deficiencies in the rigid rotor harmonic oscillator treatment can be corrected. Vibrational scaling factors can also be applied by automatic detection of the level of theory and basis set. Absolute and relative thermochemical values are output to text and graphical plots in seconds. GoodVibes provides a transparent and reproducible way to process raw computational data into publication-quality tables and figures without the use of spreadsheets.",
"keywords": [
"GoodVibes",
"thermochemistry",
"quasi-harmonic",
"quasi-RRHO",
"automated thermodynamic calculations"
],
"content": "Introduction\n\nQuantum chemistry software packages implement various levels of theory and basis sets, so-called model chemistries, that can be used to optimize molecular geometries and compute ground state vibrational modes. Statistical mechanical expressions using these vibrational frequencies, along with other contributing terms to the partition function, are used to obtain the enthalpy, entropy and Gibbs energy values required to understand, validate or predict experimental observations. GoodVibes was developed to address several challenges faced by practitioners of computational thermochemistry.\n\nFirstly, the rigid-rotor harmonic oscillator (RRHO) model is routinely used to obtain vibrational entropic contributions and is the default for most electronic structure packages. However, the harmonic approximation fails to accurately describe low frequency modes and alternative models may be more appropriate1–3. Corrections to the RRHO model may be theoretically desirable, but the absence of practical and accessible tools to implement such corrections has limited their widespread adoption. Here we present and detail the use of the program GoodVibes4, a Python-based project used for obtaining thermochemical values while applying corrections using quasi-harmonic approximations, alongside other corrections relevant to the overestimation of the zero-point energy, multi-conformer ensembles, and standard concentrations.\n\nSecondly, computational chemistry projects often combine results from different software. For example, geometries may be optimized with one program and the energies evaluated with another. Complex spreadsheets are frequently used to process these results and to prepare figures and manuscripts. However, errors in spreadsheets are commonplace and may be hard to detect. For this reason, GoodVibes allows the combination of data from multiple program outputs as part of the same project. Our group has utilized this program in informatics and organic mechanistic studies5, however, GoodVibes is not limited to a specific area of chemistry and has been used in published studies by more than 30 different research groups. As an example, previous studies in organic catalysis and mechanism, photocatalysis, and inorganic structure characterization have made use of this program package to process and make corrections to thermochemical data6. Figure 1 details an overview of the GoodVibes workflow from input data to the various outputs. Input options supplied via the command line enable chemists with basic experience with Python to process a large number of computational output files and to generate publication-ready data. These data (e.g. figures, tables) can be quickly reproduced by any other user with access to the raw data and the GoodVibes code. With recent and coming changes to standards of supplying full computational outputs through open repositories (such as Zenodo and ioChem-BD7) alongside publications, GoodVibes allows for complete transparency in how reported thermochemical values were obtained.\n\nComputational chemistry output data files are parsed, and thermochemistry data is generated based on a series of user-defined physical assumptions and reaction conditions. The program outputs absolute and relative values to text and reaction profiles as plots. The execution time is seconds.\n\n\nMethods\n\nBy recomputing translational, rotational, vibrational and electronic partition functions from the data generated by quantum chemistry programs (such as vibrational frequencies, molecular mass, etc.), thermochemistry can be calculated in GoodVibes at any specified temperature or concentration/pressure. By default, these are set to 298.15 K and 1 atmosphere. A notable automated correction to the RRHO model is applied to low frequency vibrational modes. These low frequency modes (typically less than 100 cm-1) are not well approximated as harmonic and their entropy contributions tend to be overestimated. Methods to avoid this include nontrivial hindered-rotor calculations and computationally expensive anharmonic calculations, both of which become more infeasible with higher atom counts1. Both Cramer/Truhlar2 and Grimme3 have proposed simple, more widely adopted corrections, so-called quasi-harmonic approximations, formulated specifically to obtain vibrational entropies for these low frequency modes. GoodVibes will also automatically apply empirical scaling factors to computed frequencies. These corrections arise from the tendency of electronic structure calculations (e.g. with density functional theory) to overestimate vibrational frequencies relative to experiment, and hence zero-point energies. Linear scaling factors have been collated for a number of functional and basis set combinations for sets of small organic molecules. GoodVibes accesses the scaling factors compiled by the Truhlar group across several studies8, automatically detects the level of theory and basis set and scales the frequencies if there is a match.\n\nSingle point energy calculations performed at more expensive levels of theory and with larger basis sets are commonly used in combination with the thermal corrections obtained from separate calculations, often using different software packages9. A multitude of output files from different program packages along with correction applied by GoodVibes can be combined to construct a potential energy surface by using an easily interpretable YAML file that defines the elementary steps of the reaction, file definitions and formatting options.\n\nGoodVibes is a module implemented in Python, currently supported by 2.6-7 and all 3.x versions. The module requires the NumPy library (version 1.14.2 or greater), with optional importing of Matplotlib (version 2.2.4 or greater) to graph potential energy surfaces. To test the accuracy and upkeep of this coding package, a series of tests have been implemented in TravisCI, checking functionality and accuracy of the code when the master branch is updated on GitHub against target Python versions on Linux, macOS and Windows operating systems.\n\nGoodVibes is compatible with Windows 10, Linux and macOS operating systems. This software is appropriate for use with output files produced by a wide range of calculation types, including density functional theory, wave function theory, molecular mechanics, COSMO-RS solvation calculations, and semi-empirical methods. Current supported programs include Gaussian 0910, Gaussian 1611, ORCA 412 single point energy calculation files, and COSMOtherm13 COSMO-RS solvation free energy output files. GoodVibes provides an output file with tabulated thermochemical data, optionally exported as a CSV file. Cartesian coordinates of processed files can also optionally be exported in an XYZ file. Plots of energy profiles are optionally generated.\n\n\nUse case\n\nIn this section we show how GoodVibes can be used with a variety of input options and files to transform heterogeneous computational chemistry data into human-readable tabulated values and figures. In this example, 50 calculation output files (25 Gaussian geometry optimizations and vibrational frequency calculations with 25 corresponding ORCA single point calculations) are used to create the data in Table 1 and graphed in Figure 2. All raw data was taken from a 2018 study14, and is freely accessible through a Zenodo repository15. Each point in Figure 2 represents a unique conformer’s Gibbs energy. The Boltzmann-weighted values are shown as dashes connected by the curved profiles. Optimizations were done with ωB97X-D/6-31+G(d)16 implemented in Gaussian using an “ultrafine” pruned (99,590) integration grid. Considering solvent effects of the reaction, calculations were run with the SMD solvation model17 using ethanol, and the concentration of each substance was set to 1.0 M for further thermochemical calculation. GoodVibes allows for solvent media corrections to entropy based on select solvent standard state concentration18, which was applied to ethanol in this case.\n\nIncluding: single point calculations (ΔE_SPC), energy (ΔE), zero-point energy (ΔZPE), Gibbs free energy (ΔG) and quasi-harmonic corrected Gibbs free energy (qh-ΔG).\n\nThermochemistry is evaluated at a temperature of 80°C (353.15 K) in accordance with experimental conditions (the temperature assumed in the original calculations does not influence the results from GoodVibes). These values are corrected using the quasi-harmonic approximation proposed by Grimme. One conformer of intermediate II in the ‘Py’ pathway has a small imaginary frequency of 2.9 cm-1, which was “inverted” to a real value 2.9 cm-1, as done in previous works to small imaginary frequencies, typically under >i50 cm-114,19.\n\nSeparate single point energies are extracted from ORCA calculations performed at a coupled cluster level of theory with a (DZ/TZ) basis set extrapolation (DLPNO-CCSD(T), cc-pVDZ/cc-pVTZ)20, then used for calculations and added to the GoodVibes output for comparison. A potential energy surface is constructed by using Boltzmann weighted averaging of all conformers at each step in the pathway. A multi-structural correction is then applied to the resulting Gibbs free energy based on the number and energy of distinguishable conformers present for each species21. The Gibbs energy profile is constructed from options specified in the YAML file containing the reaction pathway steps, file definitions and plot formatting options. All of the output files, correction and formatting options are supplied to GoodVibes to output tabulated data and a graph of the reaction pathway from a single command:\n\n\n\nAdditional usage examples are described at GoodVibes GitHub repository, where several features have been added in response to requests from the community of users.\n\n\nConclusion\n\nGoodVibes is a Python-based tool that calculates thermochemical data from quantum mechanical calculations in a transparent and reproducible way. GoodVibes may be employed with any type of chemical structure, including organic and inorganic molecules of varying sizes as well as with single point calculations performed by differing programs. Additionally, GoodVibes contains many additional automated features that are designed to save time for researchers, allowing for the calculation of thermochemical data at any temperature or concentration, incorporating valuable and overlooked corrections to the RRHO model through quasi-harmonic and vibrational scaling factor corrections and construction of potential energy surfaces with applied corrections accounting for the accessibility of multiple conformations. For projects involving the analysis of a large number of computational chemistry output files, GoodVibes helps to prevent human errors associated with spreadsheets, and can be used to reproduce any table or figure from the raw data.\n\n\nData availability\n\nZenodo: Data Supporting GoodVibes: Automating and applying thermodynamic corrections to harmonic frequency calculations, https://doi.org/10.5281/zenodo.366284515.\n\nThis project contains data referenced in the use case.\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\nThis code has been made accessible for chemists of various levels of computational experience and is easily installed. The most recent version of our open-sourced Python package GoodVibes v3.0.1 is freely available on GitHub at https://github.com/bobbypaton/GoodVibes. GoodVibes may be installed as a Python module from the command line using either PyPI (https://pypi.org/project/goodvibes/) or Conda (https://anaconda.org/patonlab/goodvibes) using the commands:\n\n\n\nor:\n\n\n\nor, by downloading the repository from GitHub and running the following command from the extracted directory:\n\n\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.59524622\n\nLicense: MIT",
"appendix": "Acknowledgements\n\nYanfei Guan (MIT), Jaime Rodríguez-Guerra (Charité Universitätsmedizin Berlin Institut für Physiologie).\n\n\nReferences\n\n[ (a) ] Ayala PY, Schlegel HB: Identification and Treatment of Internal Rotation in Normal Mode Vibrational Analysis. J Chem Phys. 1998; 108(6): 2314–2325. Publisher Full Text\n\n[ (b) ] McClurg RB, Flagan RC, Goddard WA: The hindered rotor density-of-states interpolation function. J Chem Phys. 1997; 106(16): 6675–6680. Publisher Full Text\n\nRibeiro RF, Marenich AV, Cramer CJ, et al.: Use of solution-phase vibrational frequencies in continuum models for the free energy of solvation. J Phys Chem B. 2011; 115(49): 14556–14562. PubMed Abstract | Publisher Full Text\n\nGrimme S: Supramolecular Binding Thermodynamics by Dispersion-Corrected Density Functional Theory. Chem - A Eur J. 2012; 18(32): 9955–9964. Publisher Full Text\n\nLuchini G, Alegre-Requena JV, Fuenes-Ardoiz I, et al.: GoodVibes, Version 3.0.1. 2020. Reference Source\n\n[ (a) ] Liu B, Alegre-Requena JV, Paton RS, et al.: Unconventional Reactivity of Ethynylbenziodoxolone Reagents and Thiols: Scope and Mechanism. Chem Eur J. 2020; 26(11): 2386–2394. Publisher Full Text\n\n[ (b) ] Koniarczyk JL, Greenwood JW, Alegre-Requena JV, et al.: A Pyridine–Pyridine Cross-Coupling Reaction via Dearomatized Radical Intermediates. Angew Chemie. 2019; 131(42): 15024–15028. Publisher Full Text\n\n[ (c) ] Luchini G, Ascough DMH, Alegre-Requena JV, et al.: Data-mining the diaryl(thio)urea conformational landscape: Understanding the contrasting behavior of ureas and thioureas with quantum chemistry. Tetrahedron. 2019; 75(6): 697–702. Publisher Full Text\n\n[ (a) ] Lewis RD, Garcia-Borràs M, Chalkley MJ, et al.: Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase. Proc Natl Acad Sci. 2018; 115(28): 7308–7313. Publisher Full Text\n\n[ (b) ] Svatunek D, Houszka N, Hamlin TA, et al.: Chemoselectivity of Tertiary Azides in Strain-Promoted Alkyne-Azide Cycloadditions. Chem Eur J. 2019; 25(3): 754–758. Publisher Full Text\n\n[ (c) ] Gomes GDP, Loginova Y, Vatsadze SZ, et al.: Isonitriles as Stereoelectronic Chameleons: The Donor-Acceptor Dichotomy in Radical Additions. J Am Chem Soc. 2018; 140(43): 14272–14288. PubMed Abstract | Publisher Full Text\n\n[ (d) ] Wodrich MD, Busch M, Corminboeuf C: Expedited Screening of Active and Regioselective Catalysts for the Hydroformylation Reaction. Helv Chim Acta. 2018; 101(9): e1800107. Publisher Full Text\n\n[ (e) ] Ye J, Kalvet I, Schoenebeck F, et al.: Direct α-alkylation of primary aliphatic amines enabled by CO2 and electrostatics. Nature Chem. 2018; 10(10): 1037–1041. Publisher Full Text\n\n[ (f) ] Grayson MN: Mechanism and Origins of Stereoselectivity in the Cinchona Thiourea- and Squaramide-Catalyzed Asymmetric Michael Addition of Nitroalkanes to Enones. J Org Chem. 2017; 82(8): 4396–4401. PubMed Abstract | Publisher Full Text\n\n[ (g) ] Besora M, Vidossich P, Lledós A, et al.: Calculation of Reaction Free Energies in Solution: A Comparison of Current Approaches. J Phys Chem A. 2018; 122(5): 1392–1399. PubMed Abstract | Publisher Full Text\n\nÁlvarez-Moreno M, de Graaf C, López N, et al.: Managing the computational chemistry big data problem: the ioChem-BD platform. J Chem Inf Model. 2015; 55(1): 95–103. PubMed Abstract | Publisher Full Text\n\nAlecu IM, Zheng J, Zhao Y, et al.: Computational Thermochemistry: Scale Factor Databases and Scale Factors for Vibrational Frequencies Obtained from Electronic Model Chemistries. J Chem Theory Comput. 2010; 6(9): 2872–2887. PubMed Abstract | Publisher Full Text\n\nFeller D, Peterson KA, Hill JG: On the effectiveness of CCSD(T) complete basis set extrapolations for atomization energies. J Chem Phys. 2011; 135(4): 044102. PubMed Abstract | Publisher Full Text\n\nGaussian 09. Gaussian Inc., Wallingford CT, 2016. Reference Source\n\nGaussian 16. Gaussian Inc., Wallingford CT, 2016. Reference Source\n\nNeese F: Software update: the ORCA program system, version 4.0. Wiley Interdiscip Rev Comput Mol Sci. 2017; 8(1): e1372. Publisher Full Text\n\nKlamt A: Conductor-like Screening Model for Real Solvents: A New Approach to the Quantitative Calculation of Solvation Phenomena. J Phys Chem. 1995; 99: 2224–2235. Publisher Full Text\n\nHilton MC, Zhang X, Boyle BT, et al.: Heterobiaryl Synthesis by Contractive C – C Coupling via P(V) Intermediates. Science. 2018; 362(6416): 799–804. Publisher Full Text\n\nLuchini G, Alegre-Requena JV, Paton RS: Data Supporting GoodVibes: Automating and applying thermodynamic corrections to harmonic frequency calculations (Version 1.0). [Data set] Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3674160\n\n[ (a) ] Becke AD: Density-functional thermochemistry. V. Systematic optimization of exchange-correlation functionals. J Chem Phys. 1997; 107: 8554–8560. Publisher Full Text\n\n[ (b) ] Chai JD, Head-Gordon M: Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections. Phys Chem Chem Phys. 2008; 10(44): 6615–6620. PubMed Abstract | Publisher Full Text\n\n[ (c) ] Hehre WJ, Ditchfield R, Pople JA: Self-consistent molecular orbital methods. XII. Further extensions of gaussian-type basis sets for use in molecular orbital studies of organic molecules. J Chem Phys. 1972; 56: 2257–2261. Publisher Full Text\n\n[ (d) ] Hariharan PC, Pople JA: The influence of polarization functions on molecular orbital hydrogenation energies. Theoret chim Acta. 1973; 28: 213–222. Publisher Full Text\n\n[ (e) ] Krishnan R, Binkley JS, Seeger R, et al.: Self-consistent molecular orbital methods. XX. A basis set for correlated wave functions. J Chem Phys. 1980; 72: 650–654. Publisher Full Text\n\n[ (f) ] McLean AD, Chandler GS: Contracted Gaussian basis sets for molecular calculations. I. Second row atoms, Z=11–18. J Chem Phys. 1980; 72: 5639–5648. Publisher Full Text\n\n[ (g) ] Francl MM, Pietro WJ, Hehre WJ, et al.: Self-consistent molecular orbital methods. XXIII. A polarization-type basis set for second-row elements. J Chem Phys. 1982; 77: 3654–3665. Publisher Full Text\n\nMarenich AV, Cramer CJ, Truhlar DG: Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J Phys Chem B. 2009; 113(18): 6378–6396. PubMed Abstract | Publisher Full Text\n\nHarvey JN, Himo F, Maseras F, et al.: Scope and Challenge of Computational Methods for Studying Mechanism and Reactivity in Homogeneous Catalysis. ACS Catal. 2019; 9(8): 6803–6813. Publisher Full Text\n\n[ (a) ] Sure R, Grimme S: Comprehensive Benchmark of Association (Free) Energies of Realistic Host-Guest Complexes. J Chem Theory Comput. 2015; 11(8): 3785–3801. PubMed Abstract | Publisher Full Text\n\n[ (b) ] Liu Z, Patel C, Harvey JN, et al.: Mechanism and reactivity in the Morita-Baylis-Hillman reaction: the challenge of accurate computations. Phys Chem Chem Phys. 2017; 19(45): 30647–30657. PubMed Abstract | Publisher Full Text\n\n[ (a) ] Purvis GD III, Bartlett RJ: A Full Coupled-Cluster Singles and Doubles Model: The Inclusion of Disconnected Triples. J Chem Phys. 1982; 76(4): 1910–1918. Publisher Full Text\n\n[ (b) ] Pople JA, Head-Gordon M, Raghavachari K: Quadratic Configuration Interaction. A General Technique for Determining Electron Correlation Energies. J Chem Phys. 1987; 87: 5968–5975. Publisher Full Text\n\n[ (c) ] Riplinger C, Neese F: An efficient and near linear scaling pair natural orbital based local coupled cluster method. J Chem Phys. 2013; 138(3): 034106. PubMed Abstract | Publisher Full Text\n\n[ (d) ] Riplinger C, Sandhoefer B, Hansen A, et al.: Natural triple excitations in local coupled cluster calculations with pair natural orbitals. J Chem Phys. 2013; 139(13): 134101. PubMed Abstract | Publisher Full Text\n\n[ (e) ] Riplinger C, Pinski P, Becker U, et al.: Sparse maps-A systematic infrastructure for reduced-scaling electronic structure methods. II. Linear scaling domain based pair natural orbital coupled cluster theory. J Chem Phys. 2016; 144(2): 024109. PubMed Abstract | Publisher Full Text\n\nPlata RE, Singleton DA: A Case Study of the Mechanism of Alcohol-Mediated Morita Baylis-Hillman Reactions. the Importance of Experimental Observations. J Am Chem Soc. 2015; 137(11): 3811–3826. Publisher Full Text\n\nLuchin G, Alegre-Requena J, Rodríguez-Guerra J, et al.: bobbypaton/GoodVibes: GoodVibes v3.0.1 (Version v3.0.1). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3346166"
}
|
[
{
"id": "63186",
"date": "18 May 2020",
"name": "Robert Pollice",
"expertise": [
"Reviewer Expertise homogeneous catalysis",
"molecular interactions",
"noncovalent interactions",
"reaction mechanisms",
"chemical kinetics",
"organic electronics",
"machine learning"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper “GoodVibes: automated thermochemistry for heterogeneous computational chemistry data” by Paton et al. describes GoodVibes, an open-source program to compute thermochemical parameters from the output of popular computational chemistry software accounting for multiple well-established thermodynamic corrections developed and used for chemistry in the gas phase and in solutions. The aim is to provide a general tool to apply these corrections straightforwardly without much effort saving time for practitioners and making the corresponding results more reproducible for the entire scientific community.\nOverall, the paper is clear, well-written, and explains the rationale for developing the program. All the theoretical models implemented are described and cited appropriately. I believe that this program is a very important contribution to the field as it simplifies the process of applying thermochemical corrections greatly, which otherwise can be quite time-consuming and is, as the authors point out appropriately, error-prone due to the considerable number of calculation steps that need to be carried out. I have tested the current version of the program and both installation and usage are well-documented and straightforward for anyone with minimal python experience.\nHowever, having inspected the source code of the current version, I am concerned about the implementation, or lack thereof, of a few important thermochemical corrections as I will outline in the section “Major Issues” in more detail. Moreover, I believe that there are a few minor issues that require correction and I will outline them in the section “Minor Issues.”\nMajor issues:\nMy main concern with the current version of GoodVibes is that I believe that Grimme’s quasi-harmonic correction is not implemented correctly. In line 1922 of GoodVibes.py, the authors define the average moment of inertia as 10 ∙ 10-44 kg m2 and use this value as a global parameter for every molecule. However, I believe that this is a misinterpretation of the method by Grimme as described in reference 3 of the paper. In that paper, Grimme uses this specific value in Figure 2 merely as an illustration of a typical example to show how the mathematical shape of this correction looks. It is never stated that this value is to be used as a global parameter for all molecules. In fact, the average moments of inertia need to be calculated for every molecule at hand from the respective Cartesian coordinates of the conformer used and the masses of all the atoms. In my opinion, this issue needs to be addressed properly by implementing the calculation of the average moment of inertia of a conformer based on its Cartesian coordinates in GoodVibes. This is particularly important for large molecules with hundreds of atoms because their average moment of inertia will deviate significantly from the used global parameter.\n\nAfter close inspection of the code, I believe that, currently, GoodVibes applies the solvent correction as obtained from the output of COSMOtherm as is without any adjustments. However, it has been pointed out recently that this is (unfortunately) a common mistake as COSMOtherm uses the mole fraction reference state for pure solvent rather than the concentration reference state. Consequently, this leads to inconsistencies in the standard states used and to a systematic offset in predicted Gibbs free energies of solvation. The details of this problem and how to correct for it are described in the literature.1\n\nMoreover, I inspected the code of GoodVibes in order to find out whether a correction for the entropy of mixing of multiple species is applied. I could not find such a correction. I believe that this is an important correction, especially for reactions with changing molecularity. This correction is outlined in reference 21 of the paper, in particular in the SI of reference 21.\n\nMinor issues:\nPage 4, first paragraph: The authors mention that electronic structure calculations have a tendency to overestimate vibrational frequencies relative to experiment. In my opinion, this is misleading as the tendency to overestimate vibrational frequencies is not inherent to the electronic structure methods applied but rather inherent to the harmonic approximation of the vibrational potential energy surface. Hence, I suggest that the authors mention that specifically, possibly with an appropriate citation. For that purpose, citation 8 of the paper could be used as it addresses this issue properly.\n\nPage 4, “Operation” paragraph: In my opinion, appropriate citations should be added for the COSMO-RS method.\n\nPage 4, “Use case” first paragraph: The authors mention that in the output plots, “dashes [are] connected by the curved profiles.” In my opinion, the authors should mention explicitly what interpolation method is used to generate these curved profiles.\n\nPage 4, “Use case” second paragraph: The authors state that “in previous works” small imaginary frequencies “typically under >i50 cm-1” were inverted. From a mathematical point of view, I believe this to be imprecise notation. Natural linear ordering on the sets of neither complex nor imaginary numbers is possible. Hence, the notation “>i50 cm-1”, even though it has been used in Chemistry, should be avoided. To resolve this issue, it should be stated that the absolute of the imaginary frequency should be below 50 cm-1. In addition, the imaginary units should be added to the “small imaginary frequency of 2.9 cm-1” in the same sentence.\n\nThis is just a suggestion rather than an issue: It would be great to have the option to save the plots generated by GoodVibes in a number of formats including, in particular, formats supporting vector graphics like PDF as this would make it easier to use the graphics directly for generating high-resolution illustrations.\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": "63185",
"date": "26 May 2020",
"name": "Kjell Jorner",
"expertise": [
"Reviewer Expertise physical organic chemistry",
"reaction modelling",
"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\nPaton and co-workers describe an open source Python program, GoodVibes, for the calculation of thermochemistry from quantum-chemical calculations. The program automates a lot of the tedious work needed to process the output files and to add correction terms. Importantly, it presents a reproducible workflow for extracting and visualizing energies of reaction paths. GoodVibes is now in its third version and has over the last few years become a standard tool for computational chemists both in academia and industry. Additional functionality in the latest version includes, for example, automated calculation of diastereo- and enantioselectivity, potential energy surface plotting, and including separate single point energies.\nThis software tool article is, therefore, most welcome not only as a reference for the program but by providing more detail and references to the underlying theory as well as examples of how to use the program. In particular, the program implements many elements of good scientific software practice, such as using a Git repository with version control, a version release system, and continuous integration testing. The program is also easy to install via pip or conda.\nIt would be good if the authors could be more explicit regarding the “multi-structural correction” that is “applied based on the number and energy of distinguishable conformers present for each species\". Is this on by default, and what is the basis for distinguishing between unique conformers? Also, could you be more explicit regarding the symmetry detection and correction (“ssym” argument)? How does it detect the internal symmetries of the molecule? And why is it off by default, when it seems that most standard quantum chemistry codes (e.g. Gaussian) include at least external symmetry by default? Also, the use of “solvent media corrections” in conjunction with an implicit solvent method is strongly discouraged in ref 18 (Harvey, Himo, Maseras), and it would be good to add a note on this caveat with the inexperienced user in mind.\nI think the program is very useful as is, but here are some suggested improvements:\nMake the program work as an importable Python module and not only as a standalone script. This is especially helpful when integrating it into automated (Python-based) workflows.\n\nStoring internal data in separate files (e.g. csv) rather than in the Python source code may be more maintainable.\n\nSeparate documentation on, e.g., Read the Docs may be more accessible.\n\nDocument Python code with docstrings in NumPy or Google style.\n\nOutput of SVG or other vector graphics files for lossless figures.\n\nA quick way to support more quantum chemistry codes (at least on Linux or Mac) would be to support output from the UniMoVib program (https://github.com/zorkzou/UniMoVib).\nMinor correction:\n“Gibbs energy” -> “Gibbs free energy” (page 3/9 and other examples)\n\n“a Python-based project” -> “a Python-based program/tool” (page 3/9)\n\n“standard concentrations” -> “standard state concentrations” (page 3/9)\nAddition: After reading the review by Dr. Pollice, I agree with him that the handling of the moment of inertia for Grimme's method is not correct. There is a reference implementation of his method which can be found in the xtb Github repository: https://github.com/grimme-lab/xtb. The \"subroutine axis2\" handles the calculation of the average moment of inertia. After correcting for this in the code, I think the authors should do an analysis of how large the error is for using the erroneous moment of inertia, e.g., as a function of molecular size as Dr. Pollice points out.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-291
|
https://f1000research.com/articles/9-289/v1
|
24 Apr 20
|
{
"type": "Research Article",
"title": "Over-expression of Ki-67 as a predictor of lymph node metastasis in penile cancer patients",
"authors": [
"Syah Mirsya Warli",
"Ginanda Putra Siregar",
"Ginanda Putra Siregar"
],
"abstract": "Introduction: Penile cancer is a moderately common malignancy in developing countries. Metastasis to regional lymph nodes is an essential factor in a patient’s prognosis, as its occurrence predicts poor patient prognosis. As micro-metastasis occurs in more than 25% of cases, the need for more accessible diagnostic tools is necessary. Ki-67 is commonly used as a marker of proliferation associated with tumor grade and lymph node metastasis. Methods: Samples were taken from penile cancer patients between 2013 to 2018, in the form of formalin-fixed paraffin-embedded (FFPE) blocks were analyzed. Patient demographic data, current and pre-cancer condition, cancer staging, outcomes, and other results of adjuncts and treatment modalities were obtained from medical records. Immunohistochemistry analysis was carried out on FFPE preparations. Under 20% of nuclei stained was considered as low-expression and more than 20% of nuclei stained was considered as Ki-67 over-expression. Data processing and analysis were carried out using SPSS software. Results: In total, 48 FFPE samples were analyzed, with a mean patient age of 50.79 (±9.51 SD). For all patients, the type of pathology was squamous cell carcinoma. Node metastasis was positive in 34 patients (70.8%) and negative in 14 patients (29.2%). Statistical analysis was carried out using the Chi-Square test, resulting in a significant correlation between the expression of Ki-67 and lymph node metastasis in penile squamous cell carcinoma (p=0.045). Conclusion: Over-expression of Ki-67 were found in penile cancer patients with lymph node metastasis. Therefore, Ki-67 might be useful in predicting lymph node metastasis in penile cancer patients.",
"keywords": [
"Penile Cancer",
"Lymph Node Metastasis",
"Ki-67",
"Molecular Marker",
"IHC"
],
"content": "Introduction\n\nPenile cancer is one of the rare malignancies in men in developed countries. In the UK, there are 600 new cases each year, and the incidence rate has remained below 1 per 100 000 men over the years. Incidence rates are 2 - 3%, and the frequency is higher in old to middle-aged men, with a peak incidence in the sixth decade of life1. Penile cancer is a moderately common malignancy in the developing countries, specifically squamous cell carcinoma of penis, which accounts for 95% of all cases2,3. Risk factors for penile cancer include uncircumcision, phimosis, cigarette smoking, and human papillomavirus (HPV) infection4.\n\nExamination of primary lesions, regional lymph nodes, and distant metastasis is beneficial in determining the management of penile cancer. Moreover, regional lymph node involvement can also be an essential factor in patient prognosis, as its occurrence is a predictor of poor prognosis. Lymphatic metastasis in penile cancer follows the anatomical drainage route, which originates from the inguinal lymphatic nodes, then drains to both superficial and deep and pelvic lymphatic nodes.\n\nAs it holds an important role in predicting patient’s prognosis and survival, diagnosis and treatment of lymph node metastasis is essential. Physical examination is sometimes inaccurate and cannot be a reliable predictor. Patients who have a palpated inguinal lymph node may experience micro-metastasis, which occurs in more than 25% cases. To increase diagnostic accuracy and prevent unnecessary lymphadenectomy, several biomarkers have been investigated for easier diagnostics5.\n\nThe Ki-67 protein is a marker for predicting prognosis in cancers. The Ki-67 protein is a nuclear antigen that is expressed in the cell division phase, especially the G2/M phase and controls the timing of the mitosis process6. Because Ki-67 is expressed during the active phase of the cell cycle, it considered as a useful marker for cell division activity. Ki-67 is widely used as a proliferation marker related to tumor grade and lymph node metastasis in penile cancer7,8. In this study, we will look at the association of Ki-67 with lymph node metastasis in patients with penile cancer.\n\n\nMethods\n\nBefore penile cancer samples were analyzed, the research team informed patients and all patients consented to participate. The study was explained verbally, and written consent was obtained in the form of a patient signature. For patients who could not read or write, we obtained the signature of their family member as a witness or the patient’s finger print. Ethical clearance was obtained from Experimental Research Ethical Committee of University of Sumatera Utara (455/TGL/KEPK FK USU-RSUP HAM/2019).\n\nSamples were collected from penile cancer patients between 2013 and 2018. Research was conducted between August to November 2019. The samples were taken using total sampling of all penile cancer patients during this period. All of the samples included in this study were from men who fulfilled the eligibility criteria: penile cancer patients prior to any surgical intervention, chemotherapy, or radiotherapy for penile cancer; those who had an excisional biopsy sample; and those who agreed to participate in the study. The exclusion criteria for this study was a history of other malignancies.\n\nThe samples were collected as a part of routine medical care, and the additional analysis of Ki-67 was based on the histopathology sample availability. The samples were taken directly from the lesion of the patients using frozen section excisional biopsy. A urologist performed the excisional biopsy using a standard No. 20 blade under local anesthesia using 4 cc of 2% lidocaine HCl. After that, the samples were immediately fixed with 10% formalin buffer. After fixation, the tissue was dehydrated by soaking in alcohol from a low to a high concentration (70% → 95% → 100% → 100% → 100%). The next stage was purification by soaking tissue into a xylol solution twice, once for 60 minutes and second for 120 minutes, so that a transparent/clear tissue is obtained. The next step is impregnation of paraffin wax by infiltration of paraffin wax into the tissue so that the tissue becomes dense and stiff. The tissue containing paraffin wax is put in hot paraffin wax liquid, which is provided in small boxes. When this cassette is cooled, a paraffin block is formed, which contains the tissue inside. The next step is to cut the paraffin block containing tissue using a microtome with a tissue thickness of 4µ. Paraffin embedded tissue section was enhanced by pretreatment with citrate plus (ScyTek catalog# CPL500) or 10mM citrate buffer, pH 6.0 (ScyTec catalog# CBB500). Samples in the form of formalin-fixed paraffin-embedded (FFPE) blocks were analyzed in the Pathology Department, University of Sumatera Utara.\n\nPatient demographic data, current and pre-cancer condition, cancer staging, outcomes and other results of adjuncts and treatment modalities were obtained from medical records. Lymph node invasion had been assessed from either physical examination, or imaging modalities such as multi-slice computed tomography (MSCT) with intravenous contrast, and the outcome was extracted from medical records for analysis in this study.\n\nImmunohistochemistry examination was carried out on FFPE preparations. Every sample slice was warmed to a temperature of 58°C and afterwards, Xylol was used for deparaffinization (ethanol-xylene) 25% (I), 50% (II) and 70% (III) for 10 minutes each. The sample was then rehydrated with antibody solution (primary antibody was optimized in solution for use with the recommended Diagnostic BioSystems Detection System, catalog# A00095, and did not need further dilution). The primary KI-67 antibody used was rabbit polyclonal antibody (Diagnostic BioSystems, catalog# A00095). For fluorescent IHC staining of paraffin-embedded tissue sections using the KI-67 antibody, the antibody was incubated with the tissue overnight at 2–8 °C. This incubation regime optimizes specific binding of antibodies to tissue targets and reduces non-specific background staining. Antibody was incubated for 30 - 60 minutes at room temperature. From this step forward, samples were protected from light. After that, samples underwent heat-induced epitope retrieval using a boiler at 125°C at pH 6 and were cooled at 90°C for 2-10 minutes. Enzyme blocking was endogenously carried out using H2O2 0.3% and ethanol 95%. After that, the specimen was counterstained with hematoxylin. Samples were then dehydrated, cleaned (using Xylol I [25%], II [50%], III [70%]), and placed on a slide. Inspection was carried out visually using light microscopy by two examiners. Microscopic calculations were carried out in 10 visual fields containing a minimum of 500 cells using ImageJ version 1.509 application at 40x magnification. Expression of the Ki-67 protein was considered positive if brown overstaining appeared in the nucleus and cytoplasm of the cell. Expression in under 20% of stained nuclei was considered as low-expression while more than 20% of stained nuclei was considered as Ki-67 over-expression.\n\nData processing and analysis were carried out using SPSS Software version 25. Because the data we collected is categorical, we used the chi-square categorical test using SPSS.\n\n\nResults\n\nSamples were taken from 48 men, with a mean age of 50.79 (±9.51 SD). The pathology of all patients was squamous cell carcinoma. Ki-67 expression level was divided into two groups; more or less than 20%. Ki-67 expression ranged from 0% to 100%, with a median value of 4.0%10. From 48 samples, 30 patients (62.5%) were found to have Ki-67 expression equal to or less than 20%, while in 18 patients (37.5%), expression of Ki-67 was more than 20%. Patient’s characteristics are shown in Table 1.\n\nNode metastasis was found to be positive in 34 patients (70.8%) and negative in 14 patients (29.2%). Statistical analysis using the chi-square test resulted in a significant correlation between expression of Ki-67 and lymph node metastasis in penile squamous cell carcinoma (p=0.045) Table 2.\n\n1 Chi-Square test\n\n\nDiscussion\n\nThe prognosis of penile squamous cell carcinoma is heavily influenced by the incidence and severity of local lymph node metastasis11. It is characterized by the slow growth of the tumor. However, advanced regional tumor stages are often observed. Due to the low occurrence of penile squamous cell carcinoma, limited information on molecular markers for its occurrence is available12,13.\n\nLymphadenectomy is currently the gold standard in lymph node metastasis, and it has a strong therapeutic effect if performed early. However, there is a high complication risk associated with inguinal lymphadenectomy such as surgical site infection, flap necrosis, lymphocele formation, and lymphedema that correlate with wide excision of lymphatics, devascularization of skin flaps, and interruption of collateral vessels and lymphatics14.\n\nHence, finding a valid marker for the incidence of metastasis of the lymph node can lead to a significant improvement in penile squamous cell carcinoma therapy, especially to avoid overtreatment. In this research, we examined the association of Ki-67 labeling with related clinicopathological variables and patient survival in primary penile cancer.\n\nDuring all phases of the cell cycle except G0, Ki-67, which is a non-histone protein in the nuclear matrix, is expressed. The Ki-67 antigen can be found within the nucleus during interphase, whereas most of the protein is transferred to the chromosome surface in mitosis. The fact that the Ki-67 is majorly expressed during the active phase of the cell cycle makes it an excellent marker for evaluating a given cell population’s so-called growth fraction15.\n\nTo our knowledge, the association between penile Ki-67 expression and clinicopathological variables for squamous cell carcinoma has not been reported in the past. Studies show that there is a relationship between Ki-67 proliferation frequency and tumor dedifferentiation, nodal involvement, or progression of disease for head and neck squamous cell carcinomas. The lack of evidence for these variables could be due to too few patients16–18.\n\nSome previous studies have examined whether there is an association between the frequency of lymph node metastasis, the extent of invasion, and lymphatic and venous embolism, but with contradictory results12,19,20. Emerson et al.19 found that in patients with penile squamous cell carcinoma, the extent of stromal and vascular tumor invasion was predictive of cancer progression.\n\nHigh levels of Ki-67 expression has been reported to be of the prognostic predictors in several squamous cell carcinomas, such as the larynx or cervix uteri, but there is little data on Ki-67 expression in penile squamous cell carcinoma8,21,22. Penile cancer itself has three different sub-types histologically. Protzel et al.22 stated that there is a strong correlation between Ki-67 expression with the basaloid sub-type of penile cancer, while verrucous and warty types showed only medium or weak Ki-67 expression.\n\nStankiewicz et al.23, however, showed that in primary penile squamous cell carcinoma, high Ki-67 expression is associated with lymph node metastasis. This is consistent with previous studies that indicate a similar trend, but lack statistical significance8. With statistical analysis, a strong correlation was found between the expression of Ki-67 and metastasis of the lymph node (Chi-square p=0.005) and Ki-67 expression and remote metastasis (Chi-square p=0.026)7. These results are similar to those of oral squamous cell carcinomas and medullary thyroid carcinomas, which show a correlation between high-level Ki-67 expression and lymph node metastasis24,25. Similar to results of this study, Ki-67 was upregulated in patients with lymph node metastasis.\n\nKi-67 expression in penile squamous cell carcinoma is correlated with the metastasis of lymph nodes, so the staining of Ki-67 could help identify patients with a high risk of metastasis of the lymph node. In addition to that, a significant prognostic effect of Ki-67 on survival rates in uterine cervix carcinomas and other malignancies has been found25.\n\nData from studies suggest Ki-67 as a useful parameter for selecting patients who may benefit from inguinal lymphadenectomy, especially in conventional laparoscopic cholecystectomy, although the number of post-surgical complications in our sample is relatively small. For this relatively rare cancer, further investigation of molecular markers are required to predict tumor activity in penile cancer, using cohorts with more cases. This may be helpful in identifying patients who would benefit from inguinal lymphadenectomy, in addition to known prognostic factors such as pT stage and level. Further investigations will explain the effect of Ki-67 expression in unusual subtypes.\n\n\nData availability\n\nZenodo: Over-expression of Ki-67 as a predictor of lymph node metastasis in penile cancer patients. https://doi.org/10.5281/zenodo.369208810\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nNarayana AS, Olney LE, Loening SA, et al.: Carcinoma of the penis: analysis of 219 cases. Cancer. 1982; 49(10): 2185–91. PubMed Abstract | Publisher Full Text\n\nWerness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990; 248(4951): 76–9. PubMed Abstract | Publisher Full Text\n\nBleeker MC, Heideman DA, Snijders PJ, et al.: Penile cancer: epidemiology, pathogenesis and prevention. World J Urol. 2009; 27(2): 141–50. PubMed Abstract | Publisher Full Text\n\nFavorito LA, Nardi AC, Ronalsa M, et al.: Epidemiologic study on penile cancer in Brazil. Int Braz J Urol. 2008; 34(5): 587–91. discussion 591–3: PubMed Abstract | Publisher Full Text\n\nGraafland NM, Lam W, Leijte JA, et al.: Prognostic factors for occult inguinal lymph node involvement in penile carcinoma and assessment of the high-risk EAU subgroup: a two-institution analysis of 342 clinically node-negative patients. Eur Urol. 2010; 58(5): 742–7. PubMed Abstract | Publisher Full Text\n\nEndl E, Gerdes J: The Ki-67 protein: fascinating forms and an unknown function. Exp Cell Res. 2000; 257(2): 231–237. PubMed Abstract | Publisher Full Text\n\nProtzel C, Knoedel J, Zimmermann U, et al.: Expression of proliferation marker Ki-67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas. Histol Histopathol. 2007; 22(11): 1197–1204. PubMed Abstract | Publisher Full Text\n\nBerdjis N, Meye A, Nippgen J, et al.: Expression of Ki-67 in squamous cell carcinoma of the penis. BJU Int. 2005; 96(1): 146–148. PubMed Abstract | Publisher Full Text\n\nRasband WS: Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA. 1997–2018. Reference Source\n\nWarli SM, Siregar GP: Over-expression of Ki-67 as a predictor of lymph node metastasis in penile cancer patients [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3692088\n\nRavi T: Correlation between the extent of nodal involvement and survival following groin dissection for carcinoma of the penis. Br J Urol. 1993; 72(5 Pt 2): 817–9. PubMed Abstract | Publisher Full Text\n\nSolsona E, Iborra I, Rubio J, et al.: Prospective validation of the association of local tumour stage and grade as a predictive factor for occult lymph node micrometastasis in patients with penile carcinoma and clinically negative inguinal lymph nodes. J Urol. 2001; 165(5): 1506–1509. PubMed Abstract | Publisher Full Text\n\nKroon BK, Horenblas S, Lont AP, et al.: Patients with penile carcinoma benefit from immediate resection of clinically occult lymph node metastases. J Urol. 2005; 173(3): 816–819. PubMed Abstract | Publisher Full Text\n\nHorenblas S: Lymphadenectomy for squamous cell carcinoma of the penis.Part 2 the role and technique of lymph node dissection. BJU Int. 2001; 88(5): 473–483. PubMed Abstract | Publisher Full Text\n\nScholzen T, Gerdes J: The Ki-67 protein. from the known and the unknown. J Cell Physiol. 2000; 182(3): 311–22. PubMed Abstract | Publisher Full Text\n\nLiu M, Lawson G, Delos M, et al.: Predictive value of the fraction of cancer cells immunolabeled for proliferating cell nuclear antigen or Ki67 in biopsies of head and neck carcinomas to identify lymph node metastasis: Comparison with clinical and radiologic examinations. Head Neck. 2003; 25(4): 280–8. PubMed Abstract | Publisher Full Text\n\nWelkoborsky HJ, Hinni M, Dienes HP, et al.: Predicting recurrence and survival in patients with laryngeal cancer by means of DNA cytometry, tumour front grading, and proliferation markers. Ann Otol Rhinol Laryngol. 1995; 104(7): 503–10. PubMed Abstract | Publisher Full Text\n\nPignataro L, Capaccio P, Pruneri G, et al.: The predictive value of p53, MDM-2, cyclin D1 and Ki67 in the progression from low-grade dysplasia towards carcinoma of the larynx. J Laryngol Otol. 1998; 112(5): 455–9. PubMed Abstract | Publisher Full Text\n\nCubilla AL, Piris A, Pfannl R, et al.: Anatomic levels: important landmarks in penectomy specimens: a detailed anatomic and histologic study based on examination of 44 cases. Am J Surg Pathol. 2001; 25(8): 1091–1094. PubMed Abstract | Publisher Full Text\n\nEmerson RE, Ulbright TN, Eble JN, et al.: Prdicting cancer progression in patients with penile squamous cell carcinoma: the importance of depth of invasion and vascular invasion. Mod Pathol. 2001; 14(10): 963–8. PubMed Abstract | Publisher Full Text\n\nMedina Perez M, Valero Puerta J, Martinez Igarzabal MJ: Verrucous carcinoma of the penis with intense basal expression of Ki 67. Arch Esp Urol. 1999; 52(9): 983–985. PubMed Abstract\n\nProtzel C, Knodel JE, Zimmermann U, et al.: 1172: Strong expression of proliferation marker Ki67 predicts lymph node metastasis in patients with penile cancer. J Urol. 2004; 171 (Suppl) (4): 309. Publisher Full Text\n\nStankiewicz E, Ng M, Cuzick J, et al.: The prognostic value of Ki-67 expression in penile squamous cell carcinoma. J Clin Pathol. 2012; 65(6): 534–537. PubMed Abstract | Publisher Full Text\n\nMatsumoto M, Komiyama K, Okaue M, et al.: Predicting tumour metastasis in patients with oral cancer by means of the proliferation marker Ki67. J Oral Sci. 1999; 41(2): 53–56. PubMed Abstract | Publisher Full Text\n\nTisell LE, Oden A, Muth A, et al.: The Ki-67 index a prognostic marker in medullary thyroid carcinoma. Br J Cancer. 2003; 89(11): 2093–2097. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "69645",
"date": "19 Oct 2020",
"name": "Juan Chipollini",
"expertise": [
"Reviewer Expertise Urologic Oncology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed a retrospective, immunohistochemistry analysis of 48 penile cancer patients and the association of Ki-67 expression with lymph node (LN) status outcomes. They found Ki-67 was significantly associated with LN metastasis for these patients. Of note was that >70% of patients had already locally advanced disease so unclear if the proportion of patient with LN metastasis was based more on local histopathological risk features rather than Ki-67 expression alone. Likely an adjusted analysis (considering T stage, LVI, PNI, etc..) would be needed to further evaluate the impact of Ki-67 expression as a prognosticator of LN metastasis.\n\nIs the work 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": "102256",
"date": "04 Jan 2022",
"name": "Yudhistira Pradnyan Kloping",
"expertise": [
"Reviewer Expertise Urology",
"Oncology-Urology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for submitting this interesting article. However, there are several issues that need to be addressed before the article can be recommended for publication.\nThere are several grammatical errors that need to be revised.\n\nA few references are out of date. Please refrain from using references that are more than 7 years old, unless they are necessary. A few old references were necessary, however, there are too many in this.\n\nThe study design and sample size are massive limitations in this study. The authors have explained the limitations and future suggestions for this study, however, these should be elaborated further at the end of the discussion section.\n\nIt is too early to draw a conclusion that Ki-67 may be useful as a predictor. I suggest changing the title and conclusion in the abstract.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-289
|
https://f1000research.com/articles/9-284/v1
|
23 Apr 20
|
{
"type": "Clinical Practice Article",
"title": "Use of strictureplasty technique for surgical treatment of ileal endometriosis: a case series",
"authors": [
"Carlo Alboni",
"Ludovica Camacho Mattos",
"Fabio Facchinetti",
"Francesca Cabry",
"Francesco Serra",
"Andrea Ricciardolo",
"Mohamed Mabrouk",
"Roberta Gelmini",
"Carlo Alboni",
"Fabio Facchinetti",
"Francesca Cabry",
"Francesco Serra",
"Andrea Ricciardolo",
"Mohamed Mabrouk",
"Roberta Gelmini"
],
"abstract": "Intestinal deep infiltrating endometriosis is the most frequent extragenital localisation and its traditional surgical treatment is segmental resection of the affected tract. The need for implementing alternative techniques in the treatment of intestinal endometriosis arises from those cases of multiple ileal and recto-sigmoidal localisations, in which removing excessive lengths of intestine could lead to a higher rate of adverse events. Ileal endometriosis represents 4.7% of all intestinal localisations, often associated with multiple lesions and yet, to the best of our knowledge, there are no data on techniques other than intestinal resection for its treatment. Since its capacity to solve fibrostenotic lesions without removing centimeters of intestine, strictureplasty is widely implemented in the management of Crohn’s disease. We propose the use of strictureplasty for the treatment of ileal endometriosis. We performed surgical treatment for symptomatic deep infiltrating endometriosis in two patients with either ileal and sigmoidal localisations. We approached ileal nodules with strictureplasty technique, while sigmoidal nodules were removed by traditional segmental resection. No complications occurred and both patients are now asymptomatic after a 12 months-follow up. Therefore, strictureplasty could provide a tool to eliminate small bowel endometriosis maintaining a regular caliber of the ileal tract without modifying its length.",
"keywords": [
"ileal endometriosis",
"deep infiltrating endometriosis",
"resection",
"strictureplasty",
"obstruction"
],
"content": "Background\n\nEndometriosis is a benign estrogen-dependent disease in which endometrial-like tissue with stroma and glands is found outside the uterus. It can present in a variety of forms, classified as typical, subtle, cystic and deep infiltrating endometriosis (DIE) with deep endometriosis being mostly associated with pelvic pain and severity of symptoms1. The intestine is a frequent localisation of deep infiltrating endometriosis, with nodules most commonly affecting the recto-sigmoid tract (83.1%), the appendix (6.4%), the caecum and ileocaecal junction (4.1%), the small bowel (4.7%), and the omentum (1.7%)1. Symptoms that patients with intestinal endometriosis could experience are dysmenorrhea, rectal pain extended to the perineum, diarrhea, catamenial rectal bleeding, constipation and subocclusive symptoms, nausea and vomiting, cramps and abdominal distension; acute occlusion is rare2. Traditional surgical treatment for intestinal localisation is resection of the affected tract. However, in those cases with multiple nodules the surgeon must consider the risks related to a wide or multiple intestinal resection and, to the best of our knowledge, yet there are no data on specific techniques other than intestinal resection for ileal infiltrating endometriosis treatment3.\n\n\nTechnique and our experience\n\nTo avoid excessive removal of intestine in the treatment of ileal endometriosis, we propose the use of a technique known as strictureplasty. This technique was firstly described in 1977 by Katariya et al., who published the results of strictureplasties in a series of nine patients with multiple tubercular strictures producing symptoms of obstruction4. Then in 1982, Lee and Papaioannou published the results of strictureplasty on nine patients with Crohn’s disease and obstructive symptoms5. At present, strictureplasty is widely implemented in the treatment of Crohn’s disease for its capacity to solve multiple fibro-stenotic lesions without removing sections of the intestine. Several strictureplasty techniques can be performed depending on the length of the stricture, the most commonly used being the Heikene-Mikulicz technique6. It consists of making a longitudinal incision across the obstructed area extending into normal bowel on the antimesenteric side, creating an enterotomy which is sutured transversely7 (Figure 1).\n\na. Identification of the nodule/s by laparoscopic visualization and evaluation of the ileal wall by palpation once the ileal loop is exteriorized from the abdomen; b. Removal of the nodule lancing ileal wall in the shape of a lozenge with a longitudinal axis, with the mucosal sparing option; c. Transverse suture (interrupted stitches, absorbable suture, 3-0) to reduce the risk of ileal caliber reduction during healing process.\n\nIn clinical practice and in literature ileal lesions are surgically removed in different ways but, to the best of our knowledge, no technique is standardized apart from segmental resection. Herein, we performed and standardized a technique which is already fully consolidated in general surgery for the treatment of diseases which produce ileal lesions similar to those produced by endometriosis. No devices other than common surgical instrumentation have been used. The decision to use this technique was taken with the intention to treat the patients described below, and not with the aim of testing the procedures performed. Therefore, as a common clinical practice in our institution in general surgery, there was no need for consultation of IRB for approval for implementing the procedure that we describe.\n\nWe present our experience on two patients with either sigmoidal endometriosis and ileal localisations.\n\n\nCase 1\n\nA caucasian 36-year old cashier was followed at the Endometriosis referral Centre of Policlinico di Modena for deep infiltrating endometriosis which caused light dysmenorrhea, dyspareunia and heavy dyschezia. She was diagnosed with transvaginal ultrasound to have a single sigmoid localisation of endometriosis of 3 centimeters in longitudinal diameter, with partial infiltration of the sigmoid wall. Surgery was planned to remove endometriosis localisations and the patient was put on the waiting list. On June 2018, after 4 months from the first examination, she presented at Gynaecologic Emergency Department for heavy abdominal pain, dysmenorrhea and constipation from 3 days. She underwent plain abdominal radiograph which showed multiple air-fluid levels and an abdominal computed tomography (CT) showing dilation of the proximal tract of the small bowel with no clear sign of obstruction. The patient was diagnosed with suspected intestinal occlusion. Conservative treatment with fasting and hydration has been established but in the proceed12 hours the patient conditions declined with worsening of symptoms. An urgent laparoscopy was performed. During the intervention, the patient was found to have three ileal lesions suggestive of endometriosis localisation as well as the known sigmoid localisation. The mean diameter of the ileal lesions was 5 millimeters and they were all placed on the antimesenteric side of the ileal wall, from 20 to 50 centimeters from the ileo-cecal junction, causing partial stricture of that portion of the intestine. The surgical approach has been decided together in an equipe of expert gynaecologists and general surgeons. Ileal nodules have been approached by removing the trocar from the right lateral laparoscopic access and widening it to five centimeters, therein exteriorizing the interested ileal tract. The ileal nodules have been completely removed by lancing the ileal wall in the shape of a lozenge with its major axis longitudinal to the intestine itself, sparing the mucosal layer of the intestine which was not involved in the lesion and therefore not creating an enterotomy in the wall of the strictured tract. Afterwards, the ileal wall was closed transversely with a single layer, interrupted stitches, monofilament absorbable suture, 3-0. Eventually the ileum was re-inserted in the abdominal cavity through the enlarged lateral access. The same technique was performed for each of the three ileal nodules. In such a manner, it was possible to radically remove all endometriosis lesions maintaining a regular caliber of the ileal tract and without modifying its length. The intervention has been completed by resecting the sigmoid nodule with a traditional technique with Knight-Griffen8 anastomosis.\n\nPost-operative period was uneventful: the patient developed no fever or any sign of infection; she started a light diet on the second day after surgery and underwent complete re-canalisation on the fourth day after surgery. She was discharged on the fifth day after surgery with a regular diet and the prescription of Leuproreline acetate 3.75 mg, one injection per month for three months, followed by continuous combined oral contraceptive (Nomegestrol acetate 2.5 mg/Estradiol 1.5 mg). One month after surgery the patient came for a post-operative examination: she indicated no symptoms and regular alvum; no sign of relapse of the disease was found on sonographic evaluation. No long-term complications occurred in 12 months of clinical follow up, in terms of intestinal dehiscence, surgical site infection, weight loss, absorption discrepancies or relapse of the disease.\n\n\nCase 2\n\nOn January 2019 a caucasian 36-year old school teacher came to the attention of the Endometriosis referral Centre of Policlinico di Modena for consultation. She presented with chronic pelvic pain, catamenial dyspareunia, cramps and abdominal distension. After accurate history collecting, physical examination and sonographic evaluation were performed the patient was diagnosed with suspected right ovarian endometriosis, recto-sigmoidal endometriotic localisation of 4 centimeters with complete infiltration of the bowel wall and thickening of right utero-sacral ligament. Surgery was planned and in the meantime the patient was prescribed with combined oral contraceptive (Nomegestrol acetate 2.5 mg/Estradiol 1.5 mg) on a continuous regimen. After four months the patient was called in for the surgery; at that time, she indicated improvements for all symptoms with the exeption of abdominal distension, which remained constant and occasionally associated with vomiting. An operative laparoscopy was performed and all endometriotic lesions suspected on sonographic evaluation were confirmed. Moreover, a single hard nodule of ten millimeters in diameter was found on the antimesenteric surface of the ileum, approximately at 10 centimeters from ileo-cecal junction, causing a thickened stricture of the intestine, supposedly the cause of patient’s vomiting and abdominal distension. All lesions were removed, performing extensive adhesiolysis, right ovarian cystectomy, removal of right utero-sacral ligament and segmental resection of the affected recto-sigmoidal tract with a traditional technique with Knight-Griffen anastomosis. The ileal nodule was approached using strictureplasty technique: the affected ileal tract was temporarily extracted from the abdomen from the widened right lateral access; the nodule was removed from the ileal wall in the shape of a lozenge with its major axis longitudinal to the intestine; ileal mucosa was macroscopically disease-free and therefore it has been spared. A single layer suture with interrupted stitches in monofilament absorbable suture, 3-0, was performed transversely to the major axis of the incision. Eventually, the ileum was completely disease free and showed a regular caliber along the whole tract.\n\nPost-operative course was regular: fasting was stopped after two days and a light diet was commenced; complete canalization occurred on the fourth day and the patient was discharged on the sixth day after surgery. She was prescribed with a three-months course of Leuproreline acetate 3.75 mg, one injection per month, followed by continuous combined oral contraceptive pill (Nomegestrol acetate 2.5 mg/Estradiol 1.5 mg). One month after surgery, the patient was completely recovered and asymptomatic. Further follow-up examinations have been performed at 3, 8 and 12 months after surgery and the patient indicated no symptoms. She is still following the prescribed therapy with no side effects. No long-term complications occurred.\n\n\nDiscussion\n\nGiven the significant impact on the quality of life, the young average age of the patients affected by endometriosis and the need to restore a physiologic condition when treating this disease, intestinal endometriosis is far from being an easy challenge. The most common surgical treatment for intestinal localisations is segmental resection and yet, to the best of our knowledge, there is no standardized alternative for the surgical treatment of ileal endometriosis, neither laparotomic nor laparoscopic. In those cases of multiple infiltrating nodules, the decision of the type of surgical strategy needs to take into account the risk of removing excessive lengths of the intestine. This assumption conveys the necessity of implementing intestine-sparing alternative techniques in the surgical treatment of these localisations of endometriosis, thus to reduce the risk of adverse events such as short bowel syndrome9, anastomotic dehiscence, bowel obstruction and reduction in quality of life. As a matter of fact, strictureplasty is an effective and widely applied surgical technique developed as an alternative to segmental resection for patients at risk for short bowel syndrome.\n\nReporting these two cases, the authors propose strictureplasty as an option in the surgical treatment of deep infiltrating endometriosis with multiple intestinal nodules and ileal localisations. Heikene-Mikulicz technique seems to be appropriate for removing endometriosis lesions without reducing the length and caliber of the ileum. A specific strength of the procedure described is the possibility of sparing the mucosal layer of the intestine in those cases with no involvement of the mucosa, by implementing a modified Heikene-Mikulicz strictureplasty, as performed in our case series. However, due to the low incidence of small bowel endometriosis, the number of cases is still very small; therefore, larger studies are mandatory to verify the efficacy and safety of this technique and longer follow up periods are necessary to define the risks of intestinal relapse and recurrence of symptoms. Moreover, the small number of cases does not allow any precise indication on the dimension and position of ileal nodules which can be approached with this technique. It seems reasonable that nodules that transversely occupy more than one half (>50%) of the ileal diameter are more safely approached with resection; however, this assumption is only a speculation based on general surgeon experience and wider implementation of the technique with systematic reporting of the lesion dimensions and depth of infiltration is needed in order to give precise indications.\n\n\nConclusion\n\nGynaecologists need to be prepared to face and treat intestinal endometriosis with multiple recto-sigmoidal and ileal localisations. In those patients with no response or contraindication to medical therapy, a surgical approach has to be cautiously planned, considering the risks and benefits of eradication of all lesions. In order to reduce the risk of small bowel syndrome and improve the radicality of the intervention, strictureplasty technique could be an alternative to segmental resection approaching ileal nodules of small dimensions. This could provide less invasive impact on intestinal function and good results in terms of symptoms improvement and risk of recurrence.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "References\n\nChapron C, Chopin N, Borghese B, et al.: Deeply infiltrating endometriosis: pathogenetic implications of the anatomical distribution. Hum Reprod. 2006; 21(7): 1839–1845. PubMed Abstract | Publisher Full Text\n\nBianchi A, Pulido L, Espin F, et al.: [Intestinal endometriosis. Current Status]. Cir Esp. 2007; 81(4): 170–176. PubMed Abstract | Publisher Full Text\n\nWorking group of ESGE, ESHRE, and WES,Keckstein J, Becker CM, et al.: Recommendations for the surgical treatment of endometriosis. Part 2: deep endometriosis. Hum Reprod Open. 2020; 2020(1): hoaa002. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatariya RN, Sood S, Rao PG, et al.: Stricture-plasty for tubercular strictures of the gastro-intestinal tract. Br J Surg. 1977; 64(7): 496–498. PubMed Abstract | Publisher Full Text\n\nLee EC, Papaioannou N: Minimal surgery for chronic obstruction in patients with extensive or universal Crohn’s disease. Ann R Coll Surg Engl. 1982; 64(4): 229–233. PubMed Abstract | Free Full Text\n\nYamamoto T, Fazio VW, Tekkis PP: Safety and efficacy of strictureplasty for Crohn’s disease: a systematic review and meta-analysis. Dis Colon Rectum. 2007; 50(11): 1968–1986. PubMed Abstract | Publisher Full Text\n\nHesham W, Kann BR: Strictureplasty. Clin Colon Rectal Surg. 2013; 26(2): 80–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffen FD, Knight CD Sr, Whitaker JM, et al.: The double stapling technique for low anterior resection. Results, modifications, and observations. Ann Surg. 1990; 211(6): 745–751; discussion 751–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVanderhoof JA, Langnas AN: Short-bowel syndrome in children and adults. Gastroenterology. 1997; 113(5): 1767–1778. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "66784",
"date": "20 Jul 2020",
"name": "Michele Vignali",
"expertise": [
"Reviewer Expertise gynecological surgery",
"laparoscopy",
"endometriosis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 with great interest the paper titled 'Use of strictureplasty technique for surgical treatment of ileal endometriosis: a case series'. The Authors explain very clearly a new technique to remove ileal endometriosis nodules. This technique, called strictureplasty, derives from the experience of general surgeons in the treatment of Crohn's disease. The Authors report two patients treated with this technique who have had excellent results without post-operative complications and recurrence after 12 months of follow up. Excellent job!\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": "127980",
"date": "25 Mar 2022",
"name": "Philippe Koninckx",
"expertise": [
"Reviewer Expertise endometriosis surgery"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript ‘Use of strictureplasty technique for surgical treatment of ileal endometriosis: a case series’ describes the use of strictureplasty for ileal endometriosis. It is an opinion paper and difficult to judge since there are little hard data.\nMy advice would be to reconsider the manuscript after a more balanced revision. Alternatively, a simultaneous comment would be necessary.\nThe fundamental problems are:\nDeep endometriosis is not defined. If deeper than 5mm is used as a definition, an important overlap exists with typical lesions. The multifocality of deep endometriosis is strongly associated with the definition.\n\nThe deep endometriosis of the sigmoid is not clearly described and the reader might wonder whether a sigmoid resection was indicated.\n\nThe lesions shown in the picture are small superficial lesions, which we used to treat superficially with a suture to reinforce the wall.\n\nIn conclusion:\nIt is nice to emphasise that a bowel resection is not needed for this type of ileum lesion, especially since they are so rare that few people have experience.\n\nTechnically it is simple destruction (depth to be debated) with a reinforcement of the wall.\n\nA similar full discussion for the sigmoid is much more difficult.\n\nEssential for the discussion, is the diagnosis and definition of obstruction/stenosis of small bowel, ileum and large bowel.\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? No\n\nIs the conclusion balanced and justified on the basis of the findings? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-284
|
https://f1000research.com/articles/9-280/v1
|
23 Apr 20
|
{
"type": "Brief Report",
"title": "Expression and prognostic significance of MT1 isoforms in clear cell renal cell carcinoma",
"authors": [
"Xiaohua Niu",
"Ling Deng",
"Chaoming Tang",
"Dexiang Zhuo",
"Xiaohua Niu",
"Ling Deng",
"Chaoming Tang"
],
"abstract": "Metallothioneins (MTs), a family of low-weight cysteine-rich proteins, play key roles in tumor biology, such as proliferation, differentiation, apoptosis, and drug resistance. Clinical studies have demonstrated that deregulation of MTs in various types of solid cancers. However, a comprehensive overview of MT1 isoforms expression and clinical relevance in clear cell renal cell cancer (ccRCC) is lacking. The present study explored mRNA expression levels and prognostic values of MT1 isoforms in ccRCC tissues using The Cancer Genome Atlas (TCGA), Gene Expression Profiling and Interactive Analysis (GEPIA) and Oncomine database. The study observed that mRNA expression levels of six members of MT1 isoforms decreased in renal cancer tumor tissues compared with normal tissues. We further found that high-expression of MT1G, MT1H, MT1F and MT1X was related with poor overall survival time in ccRCC patients and high-expression of MT1G, MT1F and MT1X were inversely associated with disease-free survival time in ccRCC patients. Based on the correlation analysis, MT1G was identified to be co-expressed with MT1H and MT1F in ccRCC tissues. These findings suggested that MT1 isoforms mRNA may serve as diagnostic and prognostic markers for ccRCC.",
"keywords": [
"Metallothionein",
"ccRCC",
"TCGA",
"Oncomine，Bioinformatics"
],
"content": "Introduction\n\nClear cell renal cell carcinoma (ccRCC) is the most common type of renal tumor, which approximately constitutes 3% of adult malignancies and accounts for 90% of all renal malignancies1,2. In recent years, the understanding of the genetics and biology of RCC had been improved dramatically, however, the mortality for patients with advanced or metastatic ccRCC is still high3–5. Therefore, it is essential to find new biomarkers to improve the diagnosis and prognosis of ccRCC patients.\n\nMetallothioneins (MTs) are a family of cysteine-rich proteins. Their molecular weight ranges from 6 to 7 kDa and they play essential roles in tumor biology such as proliferation, differentiation, apoptosis, and drug-resistance6–11. MT proteins are encoded by MT1-4 genes family located on chromosome 16q1312, among them, MT2-MT4 are encoded by a single gene, while MT1 isoforms were encoded by a set of 14 genes, which consists of eight protein coding genes (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1M and MT-1X) and six pseudogenes (MT-1C, MT-1D, MT-1I, MT-1J, MT-1K and MT-1L)13–15. The expression of MT1 isoforms vary at cellular levels and in different tissues13. Changes in MT1 isoform expression were detected in various types of human malignancies14–18. Therefore, MT1 isoform profiling had been utilized as diagnostic markers and as prognostic predictors of tumor diseases12–14. Lots of work has been performed regarding the expression and prognostic value of MT1 isoforms in ccRCC19–22. However, there was still not enough information about the expression and prognostic value of the MT1 isoforms in ccRCC. Thus, it would be of great importance to elucidate the pattern of MT1 isoform expression and evaluate their prognostic value in ccRCC for patients' benefits.\n\nThe present study aimed to explore mRNA expression status of MT1 isoforms and their prognostic significance in ccRCC. For this purpose, we firstly investigated mRNA expression levels of MT1 isoforms in human ccRCC using The Cancer Genome Atlas (TCGA), Gene Expression Profiling and Interactive Analysis (GEPIA) and Oncomine database. Secondly, prognostic value of MT1 isoforms mRNA expression in ccRCC patients was evaluated through GEPIA by analyzing clinical data from the TCGA-KIRC project. Finally, correlations of MT1G with other MT1 isoforms were conducted using the R program to show the correlation in ccRCC.\n\n\nMethods\n\nTCGA23 is a publicly available and open access platform. As a result, ethics committee approval was not required for use of the data from this platform. GEPIA (Peking University, Peking, China) was used to assess the association between gene expression and prognosis24,25. GEPIA adopts a standard processing pipeline to analyze the RNA-Seq expression data from GTEx and TCGA, which include 8,587 normal and 9,736 tumor samples. Here, we selected the MT1 family genes, including, MT1G, MT1F, MT1X, MT1H, MT1A, MT1E, MT1M, MT1L, and the cancer types (Kidney Renal Clear Cell Carcinoma; KIRC). For the gene mRNA level expression between normal and tumor tissue in ccRCC, a boxplot using disease state as a variable was graphed to calculate differential expression of MT1 isoforms. We obtained samples from TCGA and used GEPIA to analyze the connections between overall survival and disease-free survival rates with MT1 isoforms expression in KIRC. Log-rank test was used to test the difference between Kaplan-Meier survival curves. Kaplan-Meier survival analysis was performed to analyze the relationship between the expression of MT1 isoforms and the survival days of KIRC patients25. MT1s were entered into the database to obtain Kaplan-Meier survival plots. The log rank p values and hazard ratios were calculated. A p-value < 0.05 was considered statistically significant.\n\nAnalysis of MT1 isoform expression in ccRCC was performed using the Oncomine database (www.oncomine.org; Compendia Biosciences, Ann Arbor, MI, USA). Oncomine can be accessed through the Oncomine Research Edition using an institution supplied email address26. We compared expression of the MT1 isoforms in clinical specimens of tumor datasets that contain both normal and tumor tissue. We further analyzed the MTIG gene expression in the six projects which contain both normal and tumor tissues of ccRCC. The six projects were Higgins Renal, Cutcliffe Renal, Gumz Renal, Jones Renal, Beroukhim Renal, Yusenko Renal and Lenburg Renal. Data were extracted to evaluate the expression of MT1 isoforms in RCC. In the study, p = 0.05, a fold change of 2, and a gene rank in the top 10% were set as the significance thresholds. Student's t test was used to analyze the difference in the expression of MT1s in RCC.\n\nGene expression in KIRC from the TCGA dataset was obtained through the ‘cgdsr’ R package (version 3.6.2). The mRNA sequence data and corresponding clinical traits of the TCGA-KIRC project were downloaded from TCGA, which contained 537 ccRCC tumor tissues. Gene symbol annotation information was used to match probes with corresponding genes. The correlation of MT1G with other MT1 isoforms was calculated by cor function in R using Spearman correlation and visualized using ggplot2 (version 3.3.0) and ggpubr (version 0.2.5) packages in R (version 3.6.2) in R-Studio (version 1.2.5033). The code is supplied as Underlying data.\n\n\nResults\n\nTo evaluate the MT1 isoforms mRNA expression levels in ccRCC normal and tumor tissues, we firstly analyzed transcriptome data from the TCGA-KIRC project using GEPIA. We found that mRNA expression levels of six MT1 isoforms, including MT1G, MT1H, MT1F, MT1X, MT1E and MT1M were simultaneously decreased in ccRCC tumor tissues compared with normal tissues (Figure 1). The results indicated that six out of eight transcripts of MT1 isoforms were significantly downregulated in ccRCC.\n\n(A) MT1G, (B) MT1H, (C) MT1F, (D) MT1X, (E) MT1E, (F) MT1M, (G) MT1L and (H) MT1A. The criteria thresholds for datasets were logFC >1; *p<0.01.\n\nTo further confirm the expression levels of MT1 isoform mRNA in clinical samples, we screened the Oncomine database to investigate mRNA expression of MT1 isoforms between tumor and normal tissues. As shown in Figure 2A, eight MT1 isoform mRNA levels decreased in tumor tissues compared to normal tissues. In renal tumor cases, decreased mRNA expression of MT1 isoforms were detected in a total of nine datasets for MT1G, seven for MT1F, five for MT1H and MT1X, four for MT1H, four for MT1E, and two for MT1M. Taken together, these results indicate that mRNA expression levels of MT1 isoforms are downregulated in renal tumor.\n\n(A) Expressions of MT1 isoform mRNA in 20 common cancers were compared with those in corresponding normal tissues. The search criteria thresholds for datasets of cancer versus normal analysis were p-value of <0.0001, a fold change of >2, and a gene rank in the top 10%. Red signifies gene upregulated in the analyses; blue represents gene downregulated. (B) Confirmation of the MT1G decreased expression in clear cell renal cell carcinoma tumor tissues compared to normal tissues based on the Oncomine database in different projects.\n\nTo investigate the decreased expression of MT1G mRNA expression level in ccRCC tissues, we investigated the differences in mRNA level of MT1G between tumor and normal tissues in ccRCC using the Oncomine database. As shown in Figure 2B, there were six ccRCC projects containing tumor and normal tissues and all the datasets indicated that the expression levels of MT1G dramatically decreased in tumor tissues. Taken together, these results from clinical specimens demonstrate that mRNA expression levels of MT1G are significantly downregulated in ccRCC tumor tissues.\n\nTo figure out the MT1 isoform transcripts that are co-expressed with the MT1G transcript, we performed a correlation analysis between mRNA expression levels of MT1G and other MT1 isoform transcripts based on TCGA-KIRC transcriptome data (Spearman correlation shown in Figure 3). A strong correlation of mRNA expression in cancer tissues (r≥0.8) was found between MT1G and MT1H, MT1G and MT1F, a moderate correlation (r≥0.5) existed between MT1G and MT1M, MT1X or MT1E. The expression of eight MT1 isoform mRNA and clinicopathological information involved in this study are supplied in Underlying data.\n\n(A) MT1G, (B) MT1H, (C) MT1F, (D) MT1M, (E) MT1X, (F) MT1E, (G) MT1A and (H) MT1L.\n\nTo assess the value of eight MT1 isoforms in ccRCC prognosis, the relevance between MT1 isoforms and overall survival time and disease-free survival time were investigated. First, we examined the prognostic relevance of eight MT1 isoform mRNA expression in ccRCC using GEPIA survival analysis based on TCGA-KIRC clinicopathological data. The patients from the TCGA-KIRC dataset (n = 516, which have both the transcriptome data and clinicopathological data) were classified into low- and high-expression groups according to the median of the specific gene mRNA expression level. For MT1G, the clinicopathological information of different MT1G expression groups is shown in Table 1; survival differences between the low and high expression groups were detected with Kaplan-Meier survival curves. mRNA expression levels of MT1G, MT1H, MT1F and MT1X were inversely related to survival according to the Kaplan-Meier survival analysis (Figure 4). However, mRNA expression levels of MT1A, MT1E, MT1L, and MT1M were not significantly associated with the clinical outcome of ccRCC patients. We also found a shorter disease-free survival time in patients with higher mRNA expression of MT1G, MT1F and MT1X (Figure 5). To answer whether mRNA expression levels of MT1 isoforms correlated with ccRCC progression, we analyzed the relationship between MT1 isoform mRNA expression levels and tumor stage. As Figure 6 shows, MT1F and MT1X mRNA expression levels were positively associated with tumor stage. Collectively, these results indicate that high mRNA expression levels of MT1G, MT1F, and MT1X are associated with poor prognosis in ccRCC patients and that MT1F and MT1X expression is positively correlated with tumor stage.\n\n(A) MT1G, (B) MT1F, (C) MT1X, (D) MT1H, (E) MT1M, (F) MT1A, (G) MT1E and (H) MT1L. Red line represents samples with genes highly expressed and blue line for lowly expressed. HR: hazard ratio.\n\n(A) MT1G, (B) MT1F, (C) MT1X, (D) MT1H, (E) MT1M, (F) MT1A, (G) MT1E and (H) MT1L. Red line represents samples with genes highly expressed and blue line for lowly expressed. HR: hazard ratio.\n\n(A) MT1G, (B) MT1H, (C) MT1F, (D) MT1X, (E) MT1E, (F) MT1M, (G) MT1A and (H) MT1L.\n\n\nDiscussion\n\nThe primary goal of this study was to determine mRNA expression profile of MT1 isoforms in ccRCC tissues. Using data from TCGA-KIRC, we found that mRNA expression levels of MT1G, MT1H, MT1F, MT1X, MT1E and MT1M were simultaneously decreased in ccRCC tumor tissues compared with normal tissues. We further confirmed that MT1 isoform mRNA expression levels, especially MT1G, decreased in ccRCC tumor tissues by screening the Oncomine database. In addition, prognostic values of MT1 isoform mRNA expression in renal cancer patients were also assessed using GEPIA.\n\nLots of studies had demonstrated that the expression of MTs is not universal in human cancers13,14. Previous studies have shown that different MT isoform expressions were deregulated in various types of cancer11,13,27. Previous results have shown that MT isoforms are deregulated in ccRCC, hepatocellular carcinoma, prostate cancer, and papillary thyroid carcinoma6–10. In this study, we showed that MT1 isoform expression decreased in ccRCC tissues as analyzed by GEPIA based on TCGA-KIRC project and Oncomine database data, which were consistent with previous studies23,28,29. The results show that MT1 isoform expression decreased in tumor tissues, especially MT1G, which may play an important role in ccRCC development and serve as a diagnostic marker.\n\nIt’s well known that the expression of MT1 isoforms are downregulated in tumor tissues and high expression of MT1 isoforms are unfavorable prognostic characteristics and associated with advanced tumor stage13,28,29. Here, we found that MT1G, MT1F and MT1X were associated with poor prognosis in ccRCC patients. However, our data didn’t support a significant association between MT1G mRNA expression levels and renal cancer stage. In contrast, MT1F and MT1X showed positive associations between mRNA level and tumor stage, which were consistent with a previous studies that showed that MT1 isoforms are correlated with tumor stage12,18–20. The data suggested that MT1 isoforms may possibly be used as a clinical prognostic parameter.\n\nThe MT1 gene family contains MT-1A, -1B, -1E, -1F, -1G, -1H, -1M and -1X and the rest of MT-1 genes (MT-1C, -1D, -1I, -1J and -1L) are pseudogenes12,13. The expression and localization of individual MT1 isoforms and pseudogenes vary at cellular levels in individual tissues13. Based on the TCGA-KIRC transcriptome data, we couldn’t detect mRNA expression of MT1D, MT1C, MT-1I and MT1J in ccRCC, and we detected low expression of MT1B in renal cancer tissues, which meant a tissue specific expression of the MT1 isoforms. Furthermore, we confirmed that MT1G expression is strongly correlated to MT1H and MT1F and moderately correlated to MT1M, MT1X and MT1E. Considering the expression profiles of MT1 isoforms vary in different cancer tissues, further exploration of MT1 isoform expression profiles and function in ccRCC should be addressed.\n\nIn conclusion, this, to the best of our knowledge, is the first systematic study on MT1 isoform expression profiling in ccRCC and evaluation of the relationship between MT1 isoforms and clinical pathological features. However, further studies are required to understand the mechanism of downregulation of MT1 isoforms and the roles MT1 isoforms play in ccRCC, which would be of great importance to ccRCC patients.\n\n\nData availability\n\nOpen Science Framework: Expression and Prognostics Significance of MT1 isoforms in Clear Cell Renal Cell Carcinoma, https://doi.org/10.17605/OSF.IO/9XUND30.\n\nThis project contains the following underlying data:\n\n- Supplementary File 1: The clinicopathological information of the TCGA-KIRC project.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nSiegel RL, Miller KD, Jemal A: Cancer statistics, 2019 CA Cancer J Clin. 2019; 69(1): 7–34. PubMed Abstract | Publisher Full Text\n\nMotzer RJ, Jonasch E, Agarwal N, et al.: Kidney cancer, version 3.2015. J Natl Compr Canc Netw. 2015; 13(2): 151–59. PubMed Abstract | Publisher Full Text\n\nMiller KD, Nogueira L, Mariotto AB, et al.: Cancer treatment and survivorship statistics. CA Cancer J Clin. 2019; 69(5): 363–385. PubMed Abstract | Publisher Full Text\n\nPeinemann F, Unverzagt S, Hadjinicolaou AV, et al.: Immunotherapy for metastatic renal cell carcinoma: A systematic review. J Evid Based Med. 2019; 12(4): 253–62. PubMed Abstract | Publisher Full Text\n\nYang F, Wang Y, Wu J, et al.: Analysis of long non‑coding RNA expression profiles in clear cell renal cell carcinoma. Oncol Lett. 2017; 14(3): 2757–2764. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWojtczak B, Pula B, Gomulkiewicz A, et al.: Metallothionein Isoform Expression in Benign and Malignant Thyroid Lesions. Anticancer Res. 2017; 37(9): 5179–5185. PubMed Abstract | Publisher Full Text\n\nSun X, Niu X, Chen R, et al.: Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology. 2016; 64(2): 488–500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHung KC, Huang TC, Cheng CH, et al.: The Expression Profile and Prognostic Significance of Metallothionein Genes in Colorectal Cancer. Int J Mol Sci. 2019; 20(16): pii: E3849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHenrique R, Jerónimo C, Hoque MO, et al.: MT1G hypermethylation is associated with higher tumor stage in prostate cancer. Cancer Epidemiol Biomarkers Prev. 2005; 14(5): 1274–8. PubMed Abstract | Publisher Full Text\n\nZeng JD, Zhang N, Zhao GJ, et al.: MT1G is Silenced by DNA Methylation and Contributes to the Pathogenesis of Hepatocellular Carcinoma. J Cancer. 2018; 9(16): 2807–2816. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYan DW, Fan JW, Yu ZH, et al.: Downregulation of metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in coloncancer tissue. Biochim Biophys Acta. 2012; 1822(6): 918–26. PubMed Abstract | Publisher Full Text\n\nSi M, Lang J: The roles of metallothioneins in carcinogenesis. J Hematol Oncol. 2018; 11(1): 107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrizkova S, Kepinska M, Emri G, et al.: An insight into the complex roles of metallothioneins in malignant diseases with emphasis on (sub)isoforms/isoforms and epigenetics phenomena. Pharmacol Ther. 2018; 83: 90–117. PubMed Abstract | Publisher Full Text\n\nBizoń A, Jędryczko K, Milnerowicz H: The role of metallothionein in oncogenesis and cancer treatment. Postepy Hig Med Dosw (Online). 2017; 71(0): 98–109. PubMed Abstract | Publisher Full Text\n\nDziegiel P, Pula B, Kobierzycki C, et al.: Metallothioneins in Normal and Cancer Cells. Adv Anat Embryol Cell Biol. 2016; 218: 1–117. PubMed Abstract | Publisher Full Text\n\nDutsch-Wicherek M, Sikora J, Tomaszewska R: The possible biological role of metallothionein in apoptosis. Front Biosci. 2008; 13: 4029–38. PubMed Abstract | Publisher Full Text\n\nCherian MG, Jayasurya A, Bay BH, et al.: Metallothioneins in human tumors and potential roles in carcinogenesis. Mutat Res. 2003; 533(1–2): 201–209. PubMed Abstract | Publisher Full Text\n\nBrazão-Silva MT, Rodrigues MF, Eisenberg AL, et al.: Metallothionein gene expression is altered in oral cancer and may predict metastasis and patient outcomes. Histopathology. 2015; 67(3): 358–367. PubMed Abstract | Publisher Full Text\n\nMaleckaite R, Zalimas A, Bakavicius A, et al.: DNA methylation of metallothionein genes is associated with the clinical features of renal cell carcinoma. Oncol Rep. 2019; 41(6): 3535–3544. PubMed Abstract | Publisher Full Text\n\nPal D, Sharma U, Singh SK, et al.: Metallothionein gene expression in renal cell carcinoma. Indian J Urol. 2014; 30(3): 241–244. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlkamal I, Ikromov O, Tölle A, et al.: An epigenetic screen unmasks metallothioneins as putative contributors to renal cell carcinogenesis. Urol Int. 2015; 94(1): 99–110. PubMed Abstract | Publisher Full Text\n\nDalgin GS, Drever M, Williams T, et al.: Identification of novel epigenetic markers for clear cell renal cell carcinoma. J Urol. 2008; 180(3): 1126–30. PubMed Abstract | Publisher Full Text\n\nBlum A, Wang P, Zenklusen JC: SnapShot: TCGA-Analyzed Tumors. Cell. 2018; 173(2): 530. PubMed Abstract | Publisher Full Text\n\nTang Z, Li C, Kang B, et al.: GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017; 45(W1): W98–-W102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu X, Qiu B, Yi P, et al.: Overexpression of miR-206 in osteosarcoma and its associated molecular mechanisms as assessed through TCGA and GEO databases. Oncol Lett. 2020; 19(3): 1751–1758. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhodes DR, Yu J, Shanker K, et al.: ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia. 2004; 6(1): 1–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNobeyama Y, Nakagawa H: Silencing of metallothionein 1A gene in melanoma. J Dermatol Sci. 2017; 88(2): 232–237. PubMed Abstract | Publisher Full Text\n\nMitropoulos D, Kyroudi-Voulgari A, Theocharis S, et al.: Prognostic significance of metallothionein expression in renal cell carcinoma. World J Surg Oncol. 2005; 3(1): 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTüzel E, Kirkali Z, Yörükoglu K, et al.: Metallothionein expression in renal cell carcinoma: subcellular localization and prognostic significance. J Urol. 2001; 165(5): 1710–3. PubMed Abstract | Publisher Full Text\n\nniu x: Expression and Prognostics Significance of MT1 isoforms in Clear Cell Renal Cell Carcinoma. 2020. http://www.doi.org/10.17605/OSF.IO/9XUND"
}
|
[
{
"id": "142191",
"date": "13 Jul 2022",
"name": "Peng Guo",
"expertise": [
"Reviewer Expertise Cancer 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\nPreviously it was reported that DNA methylation of metallothionein (MT) genes are associated with the clinical features of renal cell carcinoma (RCC). In the present study, the authors analyzed the association between mRNA expression levels and prognostic values of MT1 isoforms in ccRCC tissues using The Cancer Genome Atlas (TCGA); Gene Expression Profiling and Interactive Analysis (GEPIA); and Oncomine database. They found that expression of MT1 isoforms decreased in ccRCC tissues, and high expression MT1 isoforms were associated with poor prognosis of ccRCC patients. These findings indicated that expression level of MT1 isoforms mRNA may serve as diagnostic and prognostic markers for ccRCC.\nMinor points:\n\nPlease give a brief introduction of the function of metallothionein (MT) genes in cancer.\n\nThe expression of MT1 isoforms decreased in ccRCC tissues, but high expression MT1 isoforms were associated with poor prognosis of ccRCC patients, and expression of MT1 isoforms increased in higher tumor stages. Does this mean that MT1 isoforms may promote ccRCC progression while their expression decreases in ccRCC? Please explain and discuss. Thus, is it possible that the protein level of MT1 isoforms increase in ccRCC? Can you confirm this?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": []
},
{
"id": "148500",
"date": "06 Sep 2022",
"name": "Leili Saeednejad Zanjani",
"expertise": [
"Reviewer Expertise Cancer stem cells",
"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\nIn this study, Niu et al. represent a critical study aimed at investigating the Expression and prognostic significance of Metallothionein (MT1) isoforms in important subtypes of renal cell carcinoma (clear cell renal cell carcinoma (ccRCC)) using bioinformatics tools such as using The Cancer Genome Atlas (TCGA), Gene Expression Profiling and Interactive Analysis (GEPIA) and Oncomine database. They found that decreased expression of mRNA levels of six members of MT1 isoforms in renal cancer tumors compared with normal tissues. They also found that increased expression of MT1G, MT1H, MT1F, and MT1X with poor overall survival (OS) in patients with ccRCC and high expression of MT1G, MT1F, and MT1X were inversely associated with disease-free survival (DSS) in ccRCC patients. The authors concluded that MT1 isoforms mRNA may serve as diagnostic and prognostic markers for ccRCC.\nIn my opinion, the experiment design and the presentation of the data in the bioinformatics sections are good. The paper is generally well-written and structured. However, the following points should be addressed by the authors:\nThe authors need to define 'the GTEx' in the introduction section for the first time - “The Genotype-Tissue Expression”.\n\nThe authors should define the abbreviations, especially in the bioinformatics terms in figure legends.\n\nAll figure legends are highly incomplete and needed to be improved. Each figure legend should have a title and then describe the results of each section briefly and separately (A to…). The authors need to describe the results briefly in all figure legends.\n\nIt is suggested the authors describe the limitation of this study at the end of the manuscript. For example not using the experimental study to confirm the results.\n\nFigure 2, Part B must be presented separately from Part A. Because the graphs are not clear and should be shown in a larger size.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-280
|
https://f1000research.com/articles/9-186/v1
|
12 Mar 20
|
{
"type": "Brief Report",
"title": "Correlation between salivary estrogen levels and oral epithelial cytokeratin 5 expression",
"authors": [
"Juni Handajani",
"Nuraini Effendi",
"Wihaskoro Sosroseno",
"Nuraini Effendi",
"Wihaskoro Sosroseno"
],
"abstract": "Background: Estrogen expression levels may be associated with age and may affect keratinization of the hard palate. Keratinized epithelium expresses cytokeratin 5 and 14 in the basal layer. The aim of this study was to determine the correlation between the levels of salivary estrogen and number of cytokeratin 5-positive oral epithelial cells. Methods: A total of 30 female subjects were recruited and divided into children, adults and elderly (N=10 per group). Salivary estrogen levels and cytokeratin 5-expressing oral epithelial cells were assessed using ELISA and immunohistological methods, respectively. Data were analyzed using ANOVA with post hoc LSD test and Pearson’s correlation coefficient. Results: The results showed that both the number of cytokeratin 5-positive cells and the level of salivary estrogen were significantly higher in adults but decreased in the elderly, as compared with those in children (p<0.05). Furthermore, the levels of salivary estrogen were significantly correlated with the number of cytokeratin 5-positive cells (r=0.815). The ANOVA result showed significance difference cytokeratin 5 expression and estrogen level (p<0.05). The post hoc LSD test revealed cytokeratin 5 expression and estrogen level to be significantly different in children, adults, and elderly participants (p<0.05). Conclusions: These results suggest that the profile of salivary estrogen and oral epithelial cell-expressed cytokeratin 5 may be positively correlated with age and depend on age.",
"keywords": [
"Cytokeratin 5",
"Epithelial cells",
"Estrogen",
"Saliva"
],
"content": "Introduction\n\nEstrogens are known to be involved in both the female and male reproductive systems, as well as both physiological and pathological regulation of cells and tissues1. Estrogens consist of three major forms, estrone, estradiol and estriol, and function via two distinct nuclear receptors, estrogen receptor (ER)-α and ER-β1. Estrogens are able to stimulate proliferation of basal epithelial cell and differentiation epithelium, leading to up-regulated epithelial keratinization2. Since ER-β is expressed on human oral epithelium3, estrogens clearly play a crucial role in both the physiology and pathology of oral epithelium. Measurement of salivary estrogen levels is, therefore, useful to detect the individual systemic or oral condition4.\n\nCytokeratins, consisting of 19 keratins which are classified into type I (acidic) and type II (basic to neutral) keratin, are basic structural components of epithelial cells5. Cytokeratin expression is abundant on oral epithelium and salivary glands during odontogenesis6,7. Along with the expression of cytokeratin 14, the palatal epithelium during palatogenesis expresses cytokeratin 58,9. Increased cytokeratin 5-positive cells in oral mucosa were also observed in traditional batik (dye) workers and might be due to continuous exposure to azo colour dyes10. Since the levels of estrogen may be associated with oral epithelial keratinization2, the aim of the present study was to assess whether the levels of salivary estrogen expression are statistically correlated with the number of cytokeratin 5-positive oral epithelial cells.\n\n\nMethods\n\nThis study was done on July to September 2016. Using Federer11, the sample size formula was as follows:\n\n(t-1) (n-1) > 15\n\nWhere t = number of groups and n = number of subjects per group. Number of groups (t) in this study was 3 so minimal number of subjects per group (n) were 8.5. In this study, the sample size was 30 participants, with the choice of 10 participants per group was a sample of convenience. We recruited subjects in person from Sleman District, Yogyakarta, Indonesia and mitigated bias by asking subjects to fill out a questionnaire. Subjects who met inclusion criteria were asked to sign informed consent.\n\nSubjects were 30 females divided into three groups, i.e., children (8–10 years old), adult (20–30 years old), and elder (>60 years old), with 10 subjects per group. The inclusion criteria were good oral hygiene status (assessed using the Oral Hygiene Index, developed by Greene and Vermillion)12 and general health, not taking medications such as steroid, phenytoin, nifedipine, cyclosporine; not wear dentures or orthodontic appliance. Subjects approval was obtained by signing an informed consent (parents of children provided written informed consent). The protocol was approved by the Ethical Committee of the Faculty of Dentistry-Universitas Gadjah Mada (No. 00696/KKEP/FKG-UGM/EC/2016).\n\nSaliva was collected from using non-stimulating method in the afternoon (16:00–18:00) for 1 minute. Saliva sample was stored in microtubes at -20°C prior to assay. Levels of salivary estrogen were measured using an ELISA kit (Catalog No. SLV-4188; DRG Salivary Estradiol, Germany) once for each participant.\n\nEpithelial cells of the hard palate mucosa were swabbed using a cytobrush, smeared on the glass slide, fixed in methanol-acetate solution (3:1) and then kept at 4°C before staining. Slides were stained using rapid, economical, acetic acid, papanicolaou (REAP) method to identify oral epithelial cells. Slide were put in 1% acetic for 10 seconds then in Harri’s hematoxylin at 60°C for 1 minute, in Orange G-6 and EA-50 for 1 minute respectively13. Cytokeratin 5-positive cells were identified using an ABC staining kit (ImmunoCruz, Santa Cruz Biotechnology, USA). Following blocking with 1.5% blocking serum in PBS for 1 hour, the slides were then incubated overnight in a humidified chamber at 4°C with mouse monoclonal anti-cytokeratin 5 antibody (Thermo Fisher Scientific Cat# MA5-15347, RRID:AB_11009375, Thermo Fisher Scientific, USA) diluted in 1:500, immunoreacted with biotinylated secondary antibodies from a solution of 75 µl normal blocking serum stock, 5 ml PBS and 25 µl biotinylated secondary antibody stock (ImmunoCruzTM, Mouse ABC Staining System Catalog No. sc-2017 Santa Cruz Biotechnology, RRID:SCR_008987, USA) at room temperature for 30 minutes, incubated with the avidin-biotin at room temperature for 30 minutes and visualized using an ABC staining system as described by the manufacturer (Catalog No. sc-2017, Santa Cruz Biotechnology, USA). The slides were then counterstained with methylene blue and viewed under a light microscope. The number of cytokeratin 5-positive cells was counted 100 epithelial cells in 10 fields per participant.\n\nData obtained on salivary estrogen level and cytokeratin 5-positive cell expression were analyzed using ANOVA and Pearson’s correlation analysis. Following ANOVA, a post hoc least significant difference test was used to analyze differences in cytokeratin 5 expression and estrogen levels in children, adults and the elderly. Analysis was performed using IBM SPSS Statistics v22. P<0.05 was considered to indicate a statistically significant difference.\n\n\nResults\n\nThe palatal epithelial cells stained by Papanicolaou and anti-cytokeratin 5 are depicted in Figure 1. The cytokeratin 5 positive staining could be observed in the cell nucleus and cytoplasm. As seen in Figure 2, salivary estrogen levels and cytokeratin 5-positive oral epithelial cell numbers were highest in adults, followed by the elderly, and then children (p<0.05). Based on Pearson’s correlation analysis, there was a positive correlation between salivary estrogen levels and the number of cytokeratin 5-positive cells (r = 0.815). Individual-level results are available as Underlying data14.\n\nPalatal epithelial cells stained by Papanicolaou (x400) (A) and immunohistologically stained for cytokeratin 5 (B) (x400).\n\nBasal cells are stained as blue color. Intermediate and superficial are stained as pink and orange color, respectively. Cytokeratin 5 protein is stained as brown color in nucleus and cytoplasm.\n\n*p<0.05 vs. the group of children at; § p<0.05 vs the group of adults at; #p<0.05 vs the group of children at; ¶p<0.05 vs the group of adults at.\n\nResults of ANOVA are displayed in Table 1. Post hoc LSD test showed significance difference all comparison both cytokeratin 5 expression and estrogen levels (Table 2)\n\n\nDiscussion\n\nThe results in the present study showed that the highest salivary estrogen levels were found in adults, followed by the elderly and then children. These results correspond with a previous report that age changes affect hormone changes produced by the body15. Almost no gonadotrophin hormone is secreted in children, hence the ovary remains inactive16. Entering adult phase, increased estrogen levels are observed during the menstruation cycle at 24 hours before ovulation. However, estrogen levels are significantly decreased in aging16,17. The number of oral epithelial cells expressing cytokeratin 5 in adults was much higher than that in children and the elderly. The exact reason for these results remains to be studied. However, it is possible that increased cytokeratin 5-positive oral epithelial cells in adults may be due to the action of estrogens, which in turn may stimulate cell divisions on the basal layer and hence keratinization5. Indeed, statistical analysis conducted in the present study indicated that increased number of cytokeratin 5-expressing oral epithelial cells is positively correlated with increased levels of salivary estrogen. That the main estrogen function is to increase cell proliferation and differentiation and tissue homeostasis1 supports the results of present study.\n\nThe implication of the present study in the physiology and pathology of oral epithelial cells is yet to be determined. Alam and colleagues demonstrated that cytokeratin 5, along with cytokeratin 14, may function to maintain cell proliferation and differentiation in the basal layer of stratified epithelia18, suggesting that increased levels of estrogen and number of cytokeratin 5-positive oral epithelial cells in adult females may be associated with promotion and maintenance of oral epithelial cell proliferation and differentiation.\n\nIn conclusion, the present study showed that the levels of salivary estrogen and the number of cytokeratin 5-positive oral epithelial cells in adult females are significantly higher than those in the children and elderly (p<0.05). The levels of salivary estrogen were strongly correlated with the number of cytokeratin 5-positive cells (r = 0.815). Therefore, the results of the present study suggest that the levels of salivary estrogen and the number of cytokeratin 5-positive oral epithelial cells may be an age-dependent phenomenon and are positively correlated.\n\n\nData availability\n\nFigshare: Result estrogen-expresi cytokeratin 5-raw.xlsx. https://doi.org/10.6084/m9.figshare.11888727.v414.\n\nThis project contains the following underlying data:\n\nResult estrogen-expresi cytokeratin 5-raw (XLSX). Salivary estrogen levels and number of cytokeratin-5-positive cells for each participant.\n\nRaw image files used for each figure (JPG).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe would like to thank Aurita Siwi Rahmawati who helped us to collect samples.\n\n\nReferences\n\nHamilton KJ, Hewitt SC, Arao Y, et al.: Estrogen hormone biology. Curr Top Dev Biol. 2017; 125: 109–146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoos RD: Minireview: Putting physiology back into estrogens' mechanism of action. Endocrinology. 2011; 152(12): 4481–4488. PubMed Abstract | Publisher Full Text\n\nVälimaa H, Savolainen S, Soukka T, et al.: Estrogen receptor-beta is the predominant estrogen receptor subtype in human oral epithelium and salivary glands. J Endocrinol. 2004; 180(1): 55–62. PubMed Abstract | Publisher Full Text\n\nLi Y, Wang D, Zeng C, et al.: Salivary metabolomics profile of patients with recurrent aphthous ulcer as revealed by liquid chromatography-tandem mass spectrometry. J Int Med Res. 2018; 46(3): 1052–1062. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoll R, Divo M, Langbein L: The human keratins: biology and pathology. Histochem Cell Biol. 2008; 129: 705–733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang Q, Yu Y, Ruan H, et al.: Morphological and functional characteristics of human gingival junctional epithelium. BMC Oral Health. 2014; 14: 30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgawa Y: Immunocytochemistry of myoepithelial cells in the salivary glands. Prog Histochem Cytochem. 2003; 38(4): 343–426. PubMed Abstract | Publisher Full Text\n\nDanescu A, Mattson M, Dool C, et al.: Analysis of human soft palate morphogenesis supports regional regulation of palatal fusion. J Anat. 2015; 227(4): 474–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarette MJ, Lane EB, Ferguson MW: Differentiation of mouse embryonic palatal epithelium in culture: selective cytokeratin expression distinguishes between oral, medial edge and nasal epithelial cells. Differentiation. 1991; 47(3): 149–161. PubMed Abstract | Publisher Full Text\n\nHandajani J, Narissi DH: Analyze of the expression of cytokeratin 5 on the epithelial cells of the buccal mucosa in batik workers. Int J Sci Res. 2016; 5(2): 510–514. Publisher Full Text\n\nFederer WT: Experimental Design, Theory and Application. New Delhi: Oxford and IBH Publ. Co. Reference Source\n\nGreene JC, Vermillion JR: The Simplified Oral Hygiene Index. J Am Dent Assoc. 1964; 68: 7–13. PubMed Abstract | Publisher Full Text\n\nRoyBiswas R, Paral CC, Dey R, et al.: Rapid Economic, Acetic Acid, Papanicolaou Stain (REAP) - Is it suitable alternative to standard PAP stain? Al Ameen J Med Sci. 2008; 1(2): 99–103. Reference Source\n\nHandajani J, Effendi N, Sosroseno W: Result estrogen-expresi cytokeratin 5-raw.xlsx. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.11888727.v4\n\nSoldin OP, Hoffman EG, Waring MA, et al.: Pediatric reference intervals for FSH, LH, estradiol, T3, free T3, cortisol, and growth hormone on the DPC IMMULITE 1000. Clin Chim Acta. 2005; 355(1–2): 205–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDevesa J, Calcedo D: The Role of Growth Hormone on Ovarian Functioning and Ovarian Angiogenesis. Front Endocrinol (Lausanne). 2019; 10: 450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalan U, Gonsalves N, Jose M, et al.: Symptomatic changes of oral mucosa during normal hormonal turnover in healthy young menstruating women. J Contemp Dent Prac. 2012; 13(2): 178–181. PubMed Abstract | Publisher Full Text\n\nAlam H, Sehgal L, Kundu ST, et al.: Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells. Mol Biol Cell. 2011; 22(21): 4068–4078. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "61298",
"date": "23 Mar 2020",
"name": "Hirohiko Okamura",
"expertise": [
"Reviewer Expertise bone metabolism",
"transcriptional regulation",
"extracellular vesicles"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “Correlation between salivary estrogen levels and oral epithelial cytokeratin 5 expression”, Handajani et al., mainly examined the relationship salivary estrogen levels and cytokeratin 5 expression in oral epithelial cells. Salivary estrogen levels in adult group was higher than that of children group, and decreased in aging. In parallel, the number of cytokeratin 5-positive oral epithelial cells was higher in adult group compared with other groups. By using statistical analysis, the authors concluded that there is a positive correlation between the levels of salivary estrogen and the number of cytokeratin 5-positive oral epithelial cells, which may be age-dependent phenomenon. The following points should be addressed for considering indexing in the journal “F1000Research\".\n\nMajor comments;\nIn fig. 1, they showed two pictures (A) and (B) with same magnification (x400). However, there are about 10 cells can be observed in (A), whereas only single cell in (B). Why? To prove the reliability of experiment (Cytokeratin 5 staining), please show the picture, in which at least 2-3 cells can be observed.\n\nAre there any differences in cytokeratin 5 expression between basal cells and intermediate and superficial cells?\n\nIn this study, the authors examined salivary estrogen levels and cytokeratin 5 expression in only female group. Why did the authors exclude male participants? Are there any reasons?\n\nEven in the highest group (adult) of cytokeratin 5, about 2 cells were positive. Other cells are all negative? How about the expressions of other types of cytokeratin?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5412",
"date": "09 Apr 2020",
"name": "Juni Handajani",
"role": "Author Response",
"response": "1. In fig. 1, they showed two pictures (A) and (B) with same magnification (x400). However, there are about 10 cells can be observed in (A), whereas only single cell in (B). Why? To prove the reliability of experiment (Cytokeratin 5 staining), please show the picture, in which at least 2-3 cells can be observed. Answer : The raw data was showed in Result estrogen-ekspresi cytokeratin 5-raw.xlsx. https://doi.org/10.6084/m9.figshare.11888727.v118. One figure showed 2-3 positive cells of cytokeratin 5. 2. Are there any differences in cytokeratin 5 expression between basal cells and intermediate and superficial cells? Answer : Cytokeratin 5 positive cell was expressed brown color, we couldn't identify cytokeratin 5 positive cell expression from basal or intermediate or superficial cell 3. In this study, the authors examined salivary estrogen levels and cytokeratin 5 expression in only female group. Why did the authors exclude male participants? Are there any reasons? Answer : Estrogen may have role in differentiation and keratinization in oral epithelial cell. Some study showed that estrogen hormone in female higher than male. 4. Even in the highest group (adult) of cytokeratin 5, about 2 cells were positive. Other cells are all negative? How about the expressions of other types of cytokeratin? Answer : We counted the number of cytokeratin 5-positive cells per 100 epithelial cells in 10 fields per participant, the other cells are negative expression. We didn't analyze other type of cytokeratin yet."
}
]
},
{
"id": "61608",
"date": "07 Apr 2020",
"name": "Euis Reni Yuslianti",
"expertise": [
"Reviewer Expertise oral biology",
"biochemistry",
"aging",
"free radical and antioxidant"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr Handajani and collagues have worked clearly and accurately presented. The study design, statistical analysis, and its interpretation is appropriate with the methods to allow replication by other researchers. This article have good implication to explain the correlation between turn over cell in females oral mucosa with salivary estrogen levels. The discussion would be better and more deep if researchers explained about the correlation between hormonal mechanism, especially estrogen, with oral mucosa cell cycles and aging in human being.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-186
|
https://f1000research.com/articles/9-278/v1
|
22 Apr 20
|
{
"type": "Opinion Article",
"title": "A community proposal to integrate structural bioinformatics activities in ELIXIR (3D-Bioinfo Community)",
"authors": [
"Christine Orengo",
"Sameer Velankar",
"Shoshana Wodak",
"Vincent Zoete",
"Alexandre M.J.J. Bonvin",
"Arne Elofsson",
"K. Anton Feenstra",
"Dietland L. Gerloff",
"Thomas Hamelryck",
"John M. Hancock",
"Manuela Helmer-Citterich",
"Adam Hospital",
"Modesto Orozco",
"Anastassis Perrakis",
"Matthias Rarey",
"Claudio Soares",
"Joel L. Sussman",
"Janet M. Thornton",
"Pierre Tuffery",
"Gabor Tusnady",
"Rikkert Wierenga",
"Tiina Salminen",
"Bohdan Schneider",
"Sameer Velankar",
"Shoshana Wodak",
"Vincent Zoete",
"Alexandre M.J.J. Bonvin",
"Arne Elofsson",
"K. Anton Feenstra",
"Dietland L. Gerloff",
"Thomas Hamelryck",
"John M. Hancock",
"Manuela Helmer-Citterich",
"Adam Hospital",
"Modesto Orozco",
"Anastassis Perrakis",
"Matthias Rarey",
"Claudio Soares",
"Joel L. Sussman",
"Janet M. Thornton",
"Pierre Tuffery",
"Gabor Tusnady",
"Rikkert Wierenga",
"Tiina Salminen"
],
"abstract": "Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community. It also provides an important link with the genomics community, as structural bioinformaticians also use the extensive sequence data to predict protein structures and their functional sites. A very broad and active community of structural bioinformaticians exists across Europe, and 3D-Bioinfo will establish formal platforms to address their needs and better integrate their activities and initiatives. Our mission will be to strengthen the ties with the structural biology research communities in Europe covering life sciences, as well as chemistry and physics and to bridge the gap between these researchers in order to fully realize the potential of structural bioinformatics. Our Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design. Our combined efforts will be of critical importance to keep the European research efforts competitive in this respect. Here we highlight the major European contributions to the field of structural bioinformatics, the most pressing challenges remaining and how Europe-wide interactions, enabled by ELIXIR and its platforms, will help in addressing these challenges and in coordinating structural bioinformatics resources across Europe. In particular, we present recent activities and future plans to consolidate an ELIXIR 3D-Bioinfo Community in structural bioinformatics and propose means to develop better links across the community. These include building new consortia, organising workshops to establish data standards and seeking community agreement on benchmark data sets and strategies. We also highlight existing and planned collaborations with other ELIXIR Communities and other European infrastructures, such as the structural biology community supported by Instruct-ERIC, with whom we have synergies and overlapping common interests.",
"keywords": [
"structural bioinformatics",
"biomolecular structure",
"protein structure",
"nucleic acids structure",
"ELIXIR",
"Instruct-ERIC"
],
"content": "List of abbreviations\n\n3D-Bioinfo: name of the ELIXIR Community of structural bioinformatics\n\nBioExcel: Center of excellence for biomolecular research\n\nBiomedinfra: authenticaton and authorisation infrastructure (ELIXIR AAI) of ELIXIR Finland\n\nCAMEO: Continuous Automated Model Evaluation\n\nCAPRI: community-wide experiment on the comparative evaluation of protein-protein docking for structure prediction\n\nCASP: critical Assessment of protein structure prediction\n\nChEMBL: a manually curated database of bioactive molecules with drug-like properties\n\nCOOT: crystallographic object-oriented toolkit, a graphics for refinement of experimental biomolecular structures\n\nCOST: (European) cooperation in science and technology\n\nEMDB: Electron microscopy data bank\n\nELIXIR: intergovernmental organisation that brings together life science resources from across Europe\n\nEM, cryo-EM: electron microscopy, cryo-electron microscopy\n\nEOSC-Hub: European Open Science Cloud\n\nEU-OPENSCREEN: integrates high-capacity screening platforms throughout Europe\n\nFAIR: data which meet principles of findability, accessibility, interoperability, and reusability\n\nFARFAR: fragment assembly of RNA with full-atom refinement\n\nFARNA,: fragment assembly of RNA\n\nInstruct, Instruct-ERIC: pan-European research infrastructure in structural biology\n\nMD: molecular dynamics\n\nmicroED: electron micro-crystallography\n\nMMB: MacroMolecularBuilder. A program suite for macromolecular modelling\n\nMX macromolecular X-ray crystallography\n\nNMR: nuclear magnetic resonance, a spectroscopic method\n\nOpenEBench: infra-structure designed to establish a continuous automated benchmarking system for bioinformatics\n\nPDB: Protein data bank\n\nPDBe-KB: Protein data bank in Europe - knowledge base\n\nPDB-Redo: procedure to optimise crystallographic structure models\n\nPhenix: software suite for the automated determination of molecular structures\n\nPHYRE: automatic fold recognition server for predicting the structure and/or function of the protein sequence\n\nProteopedia: wiki and 3D encyclopedia of proteins and other biomolecules\n\nPubchem: database of chemical molecules and their activities\n\nRefmac: program for refinement of experimental structures of biomolecules\n\nRNA Puzzles: collective experiment for blind RNA structure prediction\n\nSAXS: small angle X-ray scattering\n\nSBDD: structure-based drug design\n\nSwiss-model: structural bioinformatics web-server dedicated to homology modelling of 3D protein structures\n\nTeSS portal: ELIXIR's training portal\n\nUniProt: Universal protein esource, a comprehensive resource for protein sequence and annotation data\n\nWeb-Beagle: web server for the alignment of RNA secondary structures\n\n\nMajor European contributions in structural bioinformatics\n\nStructural bioinformatics is a well-established scientific activity, which started in the 1970s following the establishment of the Protein Data Bank1 which provides open access to macromolecular structure models. Figure 1 illustrates major themes in structural bioinformatics. Structure data can give deeper insights into the mechanism of proteins, the functions of biomolecules (proteins, nucleic acids, carbohydrates, lipids, etc.) and their interactions with each other and with chemical modulators of their functions (inhibitors, activators, co-factors, etc.). This enables the design of new experiments to study the function of macromolecules as well as rational design of proteins and drugs, to modify their function and properties.\n\nTop row – protein structure validation, protein structure comparison and classification, protein ligand interactions, nucleic acid structures, protein-protein interactions and complexes; middle row – protein structure prediction, prediction of protein interactions, protein structure dynamics; bottom row – integration of protein structure and sequence to predict functional sites and effects of genetic variations, exploiting protein structure annotations for comparative genome studies.\n\nStructure models are experimentally determined by macromolecular X-ray crystallography (MX) and small angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy (NMR), or cryo-electron microscopy (EM). The technological developments in MX in the previous decade, largely catalysed by the structural genomics initiatives and the on-going revolution in the field of cryo-EM, are expanding the volume of structural data both quantitatively and qualitatively (see Figure 2). European structural bioinformatics groups have played a crucial role in the development of methods to validate this data2,3 and the world-wide adoption of these tools by the structural biology community. They have also initiated collaborations and joint activities between structural bioinformaticians and structural biologists, expanding recently to meet the need to develop tools and validation protocols for structures determined using new techniques such as EM (cryoEM, microED) and Integrative/hybrid methods.\n\n(a) Total number of PDB entries and number of entries for each major experimental type. (b) Number of nuclear magnetic resonance (NMR) and electron microscopy (EM) entries deposited per year. (c) Number of new entries deposited each year and number of new proteins (UniProt accession) added per year.\n\nEuropean bioinformatics groups have also been at the forefront of efforts to compare protein structures and characterise their features in order to understand the underlying principles of protein structure and function4 and thereby promote both fundamental and translational research. For example, characterisation of protein binding pockets provided vital information for rational drug design. Europe also pioneered the establishment of comprehensive structure-based protein classifications5,6 giving structural insights into protein evolution and European structure-based tools have facilitated enzyme reaction mechanism studies by chemists and biochemists.\n\nAnother major European activity, the prediction of protein structures from amino acid sequences, started in the late 1980s. European groups were amongst the first to predict protein secondary and tertiary structure for soluble and membrane associated proteins. Additionally, some of the most critical contributions to building protein 3D models from structural templates of homologous proteins, happened in Europe in the 1990s7,8, together with the development of methods for assessing model quality. European bioinformatic groups have also provided key solutions for the hardest task of de novo prediction of protein spatial structures9. Furthermore, European groups have made seminal contributions to the development of methods for modelling the 3D structure of protein complexes10, a very difficult problem, which is centre stage for today’s molecular biology. These activities have been further expanded in the field of multi-scale modelling where a wide variety of experimental and bioinformatics data are integrated into the modelling process. Importantly European groups have made major contributions to initiatives assessing the performance of structure prediction and protein docking methods (see reviews 11–13).\n\nEuropean research groups contributed significantly to the field of RNA bioinformatics, setting standards in RNA structure predictions, modeling, and data format14,15. In particular, the RNA-Puzzles experiment for evaluation of RNA structure prediction methods, and a series of associated workshops have been introduced in Europe, attracting the top groups world-wide16,17,18.\n\nProtein function is strongly related to molecular recognition of small molecules such as substrates, inhibitors, or signalling compounds and many European groups have been active in this area over the last 50 years19,20,21 and remain major players in the field. Europe also has an exemplary track record in developing molecular dynamics (MD) simulation techniques and applying them to investigate dynamic properties of protein systems, functionally important conformational transitions in proteins, as well as folding and unfolding reactions22,23,24, providing crucial insight into dynamics aspects that are notoriously difficult to capture by experimental approaches.\n\nProtein structural data and functional residue annotations also inform protein engineering, another important activity with significant European representation. For instance, the discovery of canonical conformations in antibody variable domains25 spurred the development of the first methods for accurate structure prediction in antibodies26. Other biocomputational methods have been important for enzyme engineering. Such contributions by European bioinformaticians have transformed the face of protein engineering and were the basis for establishing major biotechnological companies for developing new research and clinical tools.\n\n\nMajor challenges that 3D-Bioinfo will help to address\n\nImprovements in structure prediction opens up huge possibilities including understanding the effects of disease causing mutations, and provides an essential platform for almost all future translational efforts including developing novel drugs. Furthermore, international initiatives (i.e. CASP27, CAMEO28 and CAPRI29,30 for assessment of the prediction of protein structures and complexes have driven the field by independently validating methods and highlighting innovations that increase performance. However, many challenges still exist. It remains computationally expensive to build 3D models on a proteome-wide scale. Furthermore, prediction methods are still error prone. It is therefore important to increase coverage and confidence measures by consolidating results from multiple methods. ELIXIR is already supporting some Europe-wide collaborative initiatives. For example, a recent implementation study links several major structure prediction and annotation resources (SWISS-MODEL31, PHYRE32, GenTHREADER33, Fugue34, SUPERFAMILY35, CATH-Gene3D36) with ELIXIR Core Resources, PDBe37 and InterPro38 to increase the coverage and reliability of predicted protein structure data (see Figure 3).\n\nStructural bioinformatics tools link sequence and structure data to predict protein functional sites. As for protein structure prediction, integration of data on sites predicted by different methods will increase both coverage and accuracy. In this context, new initiatives like the PDBe Knowledgebase (PDBe-KB) are integrating data from multiple European groups allowing easy access, development of meta-predictors and common benchmarking to improve accuracy. Since some disease-associated genetic variations result in modifications of protein residues in or near functional sites, these initiatives provide a natural link with the ELIXIR Human Rare Disease Community.\n\nRecent and future technological challenges of structural biology such as EM, serial crystallography, fragment screening, bio-SAXS, time-resolved structural methods, and techniques of integrated biology in general, are important areas that can be addressed by structural (3D) bioinformatics, albeit always in close collaboration with structural biology research groups. Optimal data formats, FAIRness39 of the data, interoperability of the data and software tools are serious issues that require close collaboration between structural biologists and bioinformaticians.\n\nWith regard to prediction of protein-ligand interactions, protein/drug design, and modelling of dynamic properties of proteins and their interactions, much work remains to be done in benchmarking of methods and better integration of methods and data. 3D-Bioinfo will endeavour to facilitate collaborations and new initiatives in these areas.\n\n\nGoals of 3D-BioInfo\n\nThe major goals of 3D-Bioinfo will be to increase interoperability between resources by developing and promoting data standards, integrating data where appropriate and developing robust benchmarking strategies for prediction algorithms (e.g. protein structures, complexes, ligand/drug docking). We will also develop better visualization frameworks for protein and nucleic acid structures and work closely with the structural biology community and initiatives such as Instruct-ERIC to develop improved validation metrics for nucleic acid structures, an important area, which is currently underdeveloped.\n\nThe 3D-Bioinfo major goals can be summarized as follows:\n\n• Promote and develop data standards to drive data integration\n\n• Plan the long-term sustainability for key computational tools and data resources\n\n• Drive the integration of resources and tools for analysis of structural data\n\n• Develop robust standard methods for benchmarking and validating prediction tools\n\n• Facilitate the access of tools requiring high compute power to the appropriate facilities\n\n• Improve integration of structural data with other quantitative biological data\n\n• Pool and expand the available training and outreach material in structural bioinformatics.\n\n\nLinks between 3D-Bioinfo and the wider European research environment\n\nThe structural bioinformatics community forms an indispensable interface between producers of structural data and their users. There are already several ELIXIR endorsed node resources/servers (SWISS-MODEL31, Phyre32) and Core Data Resources (PDBe37, CATH36) in 3D-Bioinfo.\n\nEuropean groups participating in 3D-Bioinfo have been involved in European networks providing derived structural data and analysis tools to biologists (e.g. InteGr8, IMPACT, Biosapiens, Instruct-ERIC, BioExcel, Biomedinfra, EU-OPENSCREEN, EOSC-Hub) and COST Actions (e.g. CA15135 - Multi-target paradigm for innovative ligand identification in the drug discovery process; CM1306 - Understanding Movement and Mechanism in Molecular Machines, COST Action BM1405 (CA17139 EUTOPIA (EUropean TOPology Interdisciplinary Action), WP3 Entangled and Self-entangled Proteins). Other international collaborations include the Protein Structure Initiative (PSI) for structural genomics. A significant fraction of the tools/resources provided by ELIXIR nodes relates to structural bioinformatics and the combined resources receive hundreds of thousands of web-accesses/month from the wider global research community.\n\nThe 3D-Bioinfo Community includes leading figures in the field of structural bioinformatics across Europe. It was initiated in March 2018 with the formation of a Steering Committee and an Executive Committee comprising representatives from 16 ELIXIR nodes. These members actively sought further participants from within their nodes, covering a wide spectrum of skills and needs (see European groups participating in 3D-Bioinfo). They are responsible for the development and sustainable operation of complex tools ranging from databases to infrastructures promoting interoperability between multiple areas of research that rely on the reusability of structural data provided by the structural biology community (e.g. as represented by Instruct-ERIC).\n\n3D-BioInfo was launched at a meeting in Basel, September 2018, with 70 participants from 15 European countries providing their input. In addition to presentations from committee members on the proposed 3D-Bioinfo Activities, representatives of the five ELIXIR platforms on data, tools, interoperability, compute and training, described their activities. John Hancock also gave a presentation as Coordinator of ELIXIR Communities and representative of the ELIXIR hub. Additionally, 19 ‘flash talks’ were given by European research groups interested in contributing to one or more of the proposed 3D-Bioinfo activities and/or to joint activities with the ELIXIR platforms. Organisers of related ELIXIR communities (proteomics, intrinsically disordered proteins and human copy number variation) gave presentations indicating possible areas of future collaboration. There were also oral contributions from representatives of other European ESFRI initiatives like Instruct-ERIC and BioExcel, again highlighting synergies and possible collaborations. Posters from many research groups, keen to participate in 3D-Bioinfo, were presented at the meeting highlighting their possible contributions and an interactive session was held allowing participants to discuss the proposed Activities and suggest future Activities. Participants also discussed ideas for interaction with the ELIXIR platforms.\n\n\nActivities and action plans prioritized by 3D-Bioinfo\n\nFollowing their formation in March 2018, the 3D-Bioinfo Executive and Steering Committees held regular conference calls to determine the highest priority areas, which would become the main Activities to be first undertaken by the community. These 3D-BioInfo Activities, outlined below, were further discussed and refined at the launch meeting, and involve multiple participating groups across the nodes.\n\nStructural Bioinformatics is well established, as the field began more than 40 years ago following the establishment of the protein structure databank in the 1970s, so most of these Activities represent mature areas of research, some of which had already received sustained node funding to develop their tools and resources. Each Activity will have the major goals, listed above, as the core of their mission. They are being coordinated via regular conference calls and are each evolving specific tasks. Below we detail, the specific thematic aims of each Activity and highlight the planned interactions with the ELIXIR platforms.\n\nCoordinator: Sameer Velankar. The Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) both follow the FAIR principles, thus enabling many niche data resources to derive added value annotations from the archived structural data, such as structure domain classifications, information on ligand- and macromolecule binding sites and effects of mutations on structure and function. However, a lack of coordination between these specialist data resources has prevented the creation of data standards and uniform data access mechanisms, consequently reducing the impact of these valuable data.\n\nThe proposed Activity will address these omissions by further developing the PDBe Knowledge Base (PDBe-KB) – a community-driven data resource for structural and functional annotations that places structural data in its biological context. PDBe-KB increases the visibility and interoperability of niche data resources by collating minimally required common structure-based annotation data in a standardized data exchange format and by enabling comparisons between specific types of annotations obtained from different software tools.\n\nThe initial focus of this Activity will include three major goals: i) continue to expand the scope of integrating known and predicted functional site annotations with PDBe-KB; ii) integration of annotations concerning the impacts of disease-associated variants on structure and function in PDBe-KB; and iii) integration of predicted protein structure data via existing model archives by establishing a federated infrastructure for access (3D-Beacons).\n\nIdentifying additional predicted or manually curated annotations that could further enrich the integrated data of PDBe-KB: The lack of data standards and fragmented nature of specialist data resources is a barrier to the FAIR principle. We will bring together the community experts to define data standards for different types of annotations and integration of these annotations using a community-driven data exchange format will facilitate finding, accessing and reusing sparse annotations in an interoperable manner. Activity I will ensure that PDBe-KB will be an effective platform for the collaborating partners to share and compare their value-added, structure-derived annotations. Furthermore, we will continue to identify gaps in coverage of the various types of annotations in PDBe-KB and bring together community experts to fill in those gaps. The workshops will also facilitate the development and testing of new methods by providing valuable, standardized benchmarking data sets.\n\nIntegration of annotations related to the impacts of disease-associated variants on structure and function: By bringing together developers of specialist data resources and scientific software tools that can provide information on the effects of amino-acid variation on structure and function, we will establish data standards to represent these data. The data standards will support integration of these annotations in PDBe-KB with particular emphasis on the effect of disease-related mutations on conformational stability. Compiling and integrating these annotations will be highly beneficial when investigating naturally occurring variations, and when used together with all the other types of integrated annotations it may facilitate development of better tools ultimately benefiting the wider biomedical research community.\n\nIntegrating predicted protein structure models (3D-Beacons): Taking advantage of the highly interconnected data of PDBe-KB will allow the transfer of valuable annotations to structural models based on sequence- or structural similarity when experimentally determined structural data is unavailable. Development of 3D-Beacons infrastructure by incorporating structural models from existing European archives (e.g. SWISS-MODEL31, Genome3D40) and other international resources (e.g. MODBASE41 and Gremlin42) will facilitate an increase in the coverage of structure data in the sequence space. The PDBe-KB data alongside the predicted structure models will potentially allow the scientific community to gain valuable insights regarding the biological context.\n\nThe focused activities will take advantage of the existing PDBe-KB infrastructure, and will include developing community-driven data standards and a uniform data access mechanism in addition to novel, portable and distributed web-based visualisation components. Activity I will build on existing collaborations involving five ELIXIR nodes (UK, Italy, Spain, Czech Republic, Belgium).\n\nCoordinator: Shoshana Wodak. Activity II is about empowering the scientific community to extend the current information on protein 3D structures, protein interactions and assemblies, and extract knowledge from this information by using computational and bioinformatics methods, and integrating biological data from different sources. Its initial focus will be on software tools and benchmark datasets for modeling the 3D structures and conformational flexibility of proteins and protein assemblies, and on community-wide benchmarking activities that advance the field. This focus will subsequently be broadened to include other areas where 3D modelling of proteins and their interactions is most impactful. The following specific aims will be pursued.\n\nExtend the content of OpenEBench by adding a knowledge base integrating software tools for modeling the 3D structure of proteins, protein complexes and assemblies. A variety of software tools are available for modeling protein structures and protein complexes by exploiting available protein sequence data and information on known structures in the Protein Data Bank (PDB). Exploiting these data successfully involves integrating the appropriate set of tools for the problem at hand. The Community will tap into OpenEBench a knowledge portal fostering benchmarking in the life sciences domain and an important component of the ELIXIR Tools Platform, with exemplary datasets of CAMEO and CAPRI already present. The extension will include workflows and guidelines to software tools and servers for modeling protein structures and complexes, based on known structures (templates) in the PDB. More specifically tools for template-based modeling of protein assemblies, and for Integration of template-based modeling of individual subunits, with protein-protein and protein-peptide docking servers developed by members of the CAPRI community. For a more in-depth understanding of the resources please refer to the CAPRI and CAMEO websites. 3D-Bioinfo will build on these efforts and take them to the next level.\n\nDevelop a set of standard freely available tools for evaluating the quality of 3D models of proteins and protein complexes. Evaluating the quality and accuracy of 3D models of proteins and protein complexes plays a crucial role in evaluating the performance of molecular modeling methods, and helping methods developers to optimize their procedures. Furthermore, to effectively compare the performance across methods, agreed upon standard quality measures and evaluation protocols are necessary, as implemented for single protein models by CAMEO modeling (3D), the CAMEO Quality Estimation (QE) category and for protein – protein complexes by the CAPRI community. Major goals will include:\n\n1) Full automation of the CAPRI quality assessment procedures for models of protein-protein and protein-peptide complexes, and larger assemblies and making them widely accessible online and for download,\n\n2) Integration of the CAPRI and CAMEO model quality assessment tools,\n\n3) Making the integrated tools available as open software through a community repository (e.g. on GitHub, building on the GitHub established by the CAPRI community).\n\nDevelop a one-stop-shop of benchmark datasets for testing and evaluating methods for generating scoring, and ranking models of protein complexes. The availability of appropriate benchmark datasets has been crucial for the development of protein modeling procedures at all levels. Protein docking benchmarks, which assemble high resolution 3D structures of selected sets of known protein complexes and their components43,44,45 as well as their experimentally measured affinities46,47, have been widely used for benchmarking methods for protein-protein docking, and for predicting and scoring protein interaction interfaces. The OpenEBench project within ELIXIR-EXCELERATE (OpenEBench.bsc.es48) has established a transparent data model that allows not only the sharing of benchmarking datasets, but also analyzing and comparing the performance of different prediction algorithms on these datasets.\n\nOpenEBench will thus foster a collection of benchmark datasets relevant to the field of modeling the 3D structure of monomeric, homo-oligomeric and heteromeric protein complexes, extending to proteins - peptide and protein - nucleic acid interactions. It will include: 1) protein docking and affinity benchmark datasets developed by members of the CAPRI community, 2) datasets comprising all the predicted models of protein assemblies submitted to CAPRI and CASP blind prediction challenges, following the examples of the Score-Set49 derived from predicted models submitted to CAPRI, 3) a dynamic benchmark for protein complexes beyond binary interactions, considering different difficulty levels (See DynBench3D;50). In addition a mechanism will be developed for users to contribute datasets51, following well-defined community-approved standards such as the mmCIF based modeling extension developed in collaboration with the RCSB within the macromolecular ModelArchive.org project.\n\nDevelop the infrastructure to manage the CAPRI challenge, in coordination with CAMEO. For CAPRI: Develop automated registration and submission procedures (including automated validation of compliance with standard format), as well as tools for accessing and navigating target information, predicted models and prediction results on the CAPRI website. Work on these tasks is currently underway at CAPRI-EBI, but further support is needed to complete it. For CAMEO: CAMEO is currently adding a new category for heteromeric complex modeling. We propose a close collaboration with CAPRI concerning the prediction and scoring applied during fully automated evaluations.\n\nDevelop a knowledge portal to user-friendly bioinformatics and computational tools for modeling conformational flexibility of proteins. Adequate modeling of conformational changes is currently a major bottleneck in protein assembly modeling. To foster progress an important first step would be to develop a knowledge portal offering the modeling community at large, workflows and guidelines to various available computational and bioinformatics tools for modeling conformational flexibility. We will use the bio.tools registry established by the ELIXIR Tools Platform as infrastructure for this. bio.tools has tags, one of which is structure prediction, but it should be possible to add more customized tags. With such tools playing an important role in modeling intrinsically unstructured proteins, collaborations with the ELIXIR Community on protein intrinsic disorder on topics of common interest will be undertaken.\n\nCoordinator: Vincent Zoete. The biological activity of biomacromolecules is often linked to the three-dimensional recognition and binding of small molecules such as substrates, activators or inhibitors. Indeed, a large fraction of drugs are ligands targeting macromolecules like enzymes, receptors, transporters or ion channels. Consequently, important efforts have been dedicated to develop computer-aided drug design (CADD) and notably structure-based drug design (SBDD) approaches over the last decades, contributing significantly to the design of small molecules of therapeutic interest.\n\nThe field of drug discovery and development will face several challenges in the future. The on-going needs in medicinal chemistry prompts a dramatic demand for new molecular entities and the exploitation of chemical spaces that are yet unexplored. Despite important progress over the last few decades, toxicity issues remain a problem for small compounds. This has to be addressed through increased specificity, but also via a better characterization of the possible targets and the anticipation of multiple off-target effects. Toxicogenomics, pharmacogenomics, and phenotypic screening data should be collected, organized and disseminated to get a clearer overview of biomacromolecule-ligand interactions, and to ultimately predict in silico the poly-pharmacology of the compounds. New target classes are also emerging, beyond the usual well-defined binding pockets, including among others the interaction of proteins with other proteins, nucleic acids, lipids or sugars. These additions in the target classes are mirrored by the use of new classes of ligands, including peptides and macrocyclic compounds. These new types of target-ligand interactions will foster the development of novel in silico approaches, which will require new algorithms and thorough evaluations of their descriptive and predictive capacities.\n\nAmong other in silico technologies, structure based drug design (SBDD) methods remain in great need of comprehensive evaluations of their performance and domain of applicability. In particular, there is a need for improved docking and scoring methods. Although protein-ligand complexes can be predicted for small ligands and almost rigid proteins, large, flexible molecules like peptides or macrocycles and proteins with flexible binding regions are still very difficult to handle. The reliable prediction of the binding free energy of a protein-ligand complex also remains a major challenge. Better methods making use of precise chemical models, modern optimization techniques and innovative scoring approaches are still much needed but must be accompanied by comprehensive and rigorous evaluations of their performance and domain of applicability. This should go hand in hand with the creation of processing pipelines for the proper use of structural data, and of standardizing tools for IO.\n\nThe efficiency of SBDD tools often depends on molecular/physicochemical properties such as the charge, polarity or size of the protein binding site and ligand, as well as on the target class of the biomacromolecule or the chemical class of the ligand. To address this, benchmark sets should clearly list and quantify these different properties for each complex with suitable descriptors, transparent for the user community. For Activity III, we plan the following goals:\n\nCreation of benchmark datasets to assess structure-based drug design tools. Benchmarking studies performed so far, including those carried out in the CSAR52 or D3R Grand Challenge53 which focus on compound series consistently measured within one lab/institute - rely on datasets of limited size and diversity, precluding large-scale, FAIR comparisons of their performance, especially as a function of ligand and binding site properties. Activity III will therefore involve:\n\n1) Building benchmark datasets, extracted from the PDB and curated, for assessing SBDD tools on a large-scale, under well-defined FAIR conditions, thereby complementing efforts such as the D3R grand challenges. A particular effort will be devoted to collecting complexes involving peptides and macromolecules.\n\n2) Quantifying different properties (e.g. charge, polarity, size, flexibility of the binding site and ligand etc.) for each entry in the benchmark, to enable evaluation of SBDD tools as a function of these properties.\n\n3) Developing links to other databases and standardizing the retrieved data to complement the information provided for each protein-ligand complex in the benchmark sets; notably, collecting information on ligands in collaboration with PDBe-KB, and associating complexes with reliable binding affinity data using databases like ChEMBL54 and Pubchem55.\n\n4) Adding information on the non-bioactive conformations of ligands to standardize the comparison of docking calculations starting from such geometries.\n\n5) Adding information on experimentally determined non-active compounds (taken e.g. from ChEMBL54) to be used as negative examples for testing virtual screening procedures.\n\nDissemination and promotion of the benchmark datasets and results. Benchmark datasets will be made publicly available by following Open Access and FAIR principles via a collaboration with the OpenEBench project within ELIXIR EXCELERATE. The latter will also allow the sharing of benchmarking results, and comparing the performance of different prediction algorithms under FAIR conditions. Preferred standardized benchmark workflows and protocols will be published, to guarantee an objective comparison of different tools. Researchers will be encouraged to evaluate their preferred sets of SBDD approaches using the above-mentioned standardized benchmarking workflows and protocols, and to report their results. We will collect the latter and provide them to the community. This activity will be of major value to Pharma and Biotech researchers, who acknowledge the importance of standard benchmarking protocols.\n\nBiomacromolecule-ligand interactions for every scientist, educational aspects. Despite significant interest in using modelling approaches among life scientists, the use of biomacromolecules structures in molecular design largely remains a domain for experts. However, complex data preparation and association could be largely automated resulting in easier-to-use software and substantially lowering the usage barrier for life scientists. By encouraging the development of tutorials guiding the application of modelling and the interpretation of the achieved results, we will endeavour to open the world of structure-based modelling to the broader life science community.\n\nCoordinator: Bohdan Schneider. The ultimate goal and vision of Activity IV is to encourage development and use of software tools to describe, analyse, annotate, and predict nucleic acid (NA) structures. The availability and sophistication of tools dealing with various hierarchies of the nucleic acid structure lag behind the tools used to explore protein structures and this situation must be remedied. In particular, standards are needed for initial model building and refinement of nucleic acid molecular structures. This task has become urgent as new techniques, including, but not limited to cryo-EM, are generating experimental data on 3D structures containing RNA and DNA molecules, such as ribosomes, spliceosomes, polymerase assemblies, and histone complexes, faster than ever before. The modelling of large nucleic acid molecules into low-resolution electron densities is particularly challenging. The RNA structural bioinformatics community has provided prototype tools, with which to model RNA and RNA-protein complexes based on experimental data, but these tools are currently not compatible with community-wide standards describing RNA and DNA conformational space and geometry at the local (nucleotide or dinucleotide) levels.\n\nTherefore, efforts need to be directed towards formulating community-accepted benchmarks that integrate the different levels of nucleic acid structure descriptions, and more generally to improving software tools that describe, analyse, annotate, and model nucleic acid structures. To enable these developments, Activity IV will focus on the following specific goals:\n\n1) Cataloguing software tools for building nucleic acid models based on their sequences alone as well as for modelling their 3D structures using experimental data, and facilitate integration of these tools.\n\n2) Coordinating the unification of the existing NA geometry standards and formulate specifications for missing standards.\n\n3) Developing benchmarks dataset for evaluating the quality of predicted or experimentally determined NA structures.\n\nTo limit the redundancy and increase the synergy between the methods, databases, web services, and other tools, we plan to continuously update the catalogue of software tools developed by the RNA tools and software consortium, extend these tools to DNA structures and enable integration of emerging tools. These efforts will build on existing ontologies while preserving consistency with new extensions.\n\nThis integration effort will require a significant level of interoperability between data exchange protocols and software, which will be implemented following FAIR principles. Dealing with software to solve, model, and refine NA structures based on the experimental data will require a close collaboration with experimentalists. Hence joining forces with Instruct-ERIC will be essential, for reaching all the stated goals, including the development of benchmarking tools and standards.\n\nWe list a few examples of steps, which could assist useful integration of the existing tools:\n\n1) Adding links from existing servers to other tools, especially those linking 2D and 3D structure prediction. The RNA Tools and Software Consortium have already recapitulated methods for RNA secondary structure prediction and to some extent RNA 3D tools. The RNA Puzzles community of RNA structural bioinformaticians16,17,18 has developed a set of tools for linking 2D and 3D structures, software for RNA 3D structure model evaluation also exists, e.g. RASP56 or MacroMoleculeBuilder, MMB57.\n\n2) Unifying the libraries of RNA/DNA dinucleotide fragments based on the analysis of experimental structures58,59 and trinucleotide fragments60. Ultimately, the community should reach a consensus on what is the meaning of “preferred”, “allowed” and “wrong” conformers in analogy with the use of these terms in the Ramachandran plot.\n\n3) Integrating and benchmarking methods dealing with RNA structures, e.g. SimRNA61, RNAComposer62 MMB57, FARNA/FARFAR63, Web-Beagle64.\n\n4) Strengthening the collaboration with developers of the main experimental nucleic acid structure determination software tools (e.g. REFMAC65, COOT66, Phenix67, PDB-REDO68) to encourage consistent handling of NAs.\n\nThe goals of Activity IV are quite ambitious, and their implementation will require close and friendly collaboration of all research teams willing to participate; including the teams involved in this Activity, and other teams active in the field. The teams not involved in Activity IV will be encouraged to join. Successful completion of the stated goals will also depend on the close collaboration of scientists grouped under other infrastructure projects in Europe and beyond. New tools, standards and benchmarks developed for NA validation will be communicated to the experimental structural biology community, mainly Instruct-ERIC but also EuroBioImaging, to ensure consistency across the different research communities.\n\nAs a part of the 3D-Bioinfo Community, we plan to hold regular meetings and workshops and web conferences, to informally discuss progress and help identify problems that can be addressed collectively. Unlike other 3D-Bioinfo Activities, Activity IV involves a relatively new community that is less well established and will require organising workshops to encourage collaboration and to enable updating of tool catalogues and ontologies. As with the other 3D-Bioinfo Activities, we will coordinate organisation of the workshops with the ELIXIR’s training portal TeSS where appropriate. The first workshop of the Activity IV is going to take place in May 2020.\n\nThe above mentioned themes will not only form the initial focus for 3D-Bioinfo but the steering committee will actively monitor the emergence of new technologies and/or new research fields relevant for bioinformatics approaches, which then can be fostered further as new activities. For example, in the field of protein design the overarching aim is to enable completely rational design of proteins with customized biological functions e.g. novel biocatalysts for Green Chemistry to meet sustainability and environmental challenges. In order to foster new developments focused courses/workshops will be organised on such topics. For example, a course or workshop on using biocomputing to understand and engineer biocatalysis is being planned. 3D-Bioinfo will continuously seek to integrate biocomputational efforts with experimental studies in order to systematically generate, test and critically assess new hypotheses on the fundamental properties of highly active enzymes and binding proteins. This would very much increase our quantitative understanding and enhance the capabilities for the rapid generation of binders, inhibitors and biocatalysts for a range of applications in research, technology and medicine.\n\n\nInteraction of 3D-Bioinfo with other research communities\n\nAll the above 3D-Bioinfo Activities will engage with the ELIXIR platforms as described below.\n\nInteroperability platform – The outcomes of our projects must be easy to discover, to access and to integrate into users’ pipelines. This necessitates the use of standardised file formats, metadata, vocabularies and identifiers. We plan to include our resources in FAIRshairing and Identifiers.org. For all Activities we will organise community workshops to develop data exchange and retrieval standards to improve compliance with FAIR principles. Participating teams will also adopt BioSchemas69, a European led initiative. For example, different community-wide standards for evaluating predicted models of proteins and protein complexes would be integrated to promote community-wide use. FAIR-ification of benchmark datasets will be undertaken. To implement the 3D-Beacons infrastructure, we will implement a common API specification for macromolecular structure data from both experimentally determined and predicted models. Where possible, the services and tools will also be linked via workflows using common workflow language to help with interoperable workflow software development.\n\nData platform – We will link key structural bioinformatics data resources to drive the use and re-use of data. For example, data from five UK based structure prediction resources have already been integrated via Genome3D, and ELIXIR implementation studies are already supporting further integration of the data in Genome3D with data from SWISS-MODEL, developed by the Swiss node. In addition, Activity I will be responsible for the integration of data on known and predicted functional sites, from a large number of participating European groups, in PDBe-KB. Information on the structural impacts of genetic variations predicted by multiple groups will also be integrated and novel visualization strategies for presenting this integrated data will be developed. These will need to clearly distinguish between experimentally known and predicted data. Activity II will be responsible for integrating various benchmark datasets on predicted protein complexes and assemblies, and on experimentally determined complexes annotated with data from other sources (in close coordination with Activity I). Activity III will also integrate benchmark datasets. For example, we will establish Open Access benchmarks sets under FAIR principles, for assessing structure-based drug design applications. This must be done in a robust, sustainable and scalable data ecosystem, allowing the use and re-use of the data, in line with the ELIXIR Data Platform goals.\n\nTools platform – The participating tools and resources will be registered in BioTools to make them discoverable and sustainable. Currently, there are several hundred tools relating to structural bioinformatics registered in BioTools covering a range of themes. Extending the repertoire of structural bioinformatics tools in BioTools will a) ensure reproducibility, b) allow scaling up by working in cloud environments, e.g. EOSC c) make tools widely available and sustainable for non-expert users. Containerization of these tools in BioContainers will support development of complex workflows. We will use the benchmarking infrastructure OpenEBench to assess tools and help methods development. For example, all tools related to Activity I will be registered in BioTools and the developers will have the opportunity to access expertise on containerization; for Activity II, these will incorporate exemplary datasets from CAMEO and CAPRI. Workflows for modeling protein conformational flexibility and for modelling protein assemblies in the context of Cryo-EM structure determination will be designed in collaboration with respectively, BioExcel and Instruct-ERIC. The development of improved tools for validation of nucleic acid models in Activity IV feeds directly into this platform and all new methods will be registered with BioTools.\n\nCompute platform – The ELIXIR Authentication & Authorisation Infrastructure (ELIXIR-AAI) which connects to other European AAI initiatives like EGI-CheckIn, INSTRACT ARIA, will be adopted to support depositions. We will link with the compute platform activities to address scalability of the underlying infrastructure for data transfer as well as data access. For example, access to the ELIXIR compute infrastructure and to the European Open Science Cloud resources (EOSC-Hub) will be explored, to enable large-scale assessment of predicted models of protein assemblies in CAPRI prediction rounds, and to disseminate software tools and web-services. Similarly, groups providing large-scale protein structure predictions and variant impacts will seek to benefit from access to the ELIXIR compute infrastructure. We will adopt ELIXIR Authentication & Authorisation Infrastructure (ELIXIR-AAI) to support deposition of ligand-protein complexes or benchmark results into our future infrastructure. We also need to provide an easy way to store and synchronise our datasets and users’ benchmarks results across ELIXIR and other e-Infrastructures.\n\nTraining platform – PDBe-KB and participating data resources will work to add training workflows to the TeSS portal. As mentioned already, ELIXIR-UK funding has already supported preliminary work on these workflows involving collaborations between multiple partners. Proteopedia, which is being developed in collaboration between the Israeli node and PDBe at the hub, will also be a valuable mechanism for training and outreach (see Figure 4). Connections with existing initiatives such as the BioExcel Knowledge Resource Center will be established. The BioExcel knowledge resource center is a repository for computational biomolecular training resources. The resources are primarily online based, such as tutorials, online courses and videos but also include face-to-face events.\n\ntRNA plays a key role in translation, the process of synthesizing proteins from amino acids. The two arms of the \"L\" shaped molecule (cartoon) are formed by the stacking of the acceptor and TΨC-stem.\n\nWith regards to protein ligand interactions, we will train researchers on the best way to retrieve and use our benchmark datasets, and promote the usage of preferred benchmark conditions. The Molecular Modeling Group of the SIB Swiss Institute of Bioinformatics will enhance the Drug Design Workshop. This is a web-based educational tool70 to introduce structure-based computer-aided drug design to the general public. This online workshop constitutes a helpful tool to introduce the concepts of structure-based drug design to young students (15−19 years old) and to the general public. It can also be used as an introductory tool for more advanced students. Several routes of enhancements will be followed, including a better visualization and analysis of the ligand-protein complex, or the selection of new protein targets.\n\nBuilding on the experience of the CAPRI community in organizing the well-attended EMBO courses on Integrative of Biomolecular Complexes (3 editions so far), Activity II will coordinate various training activities and workshops on teaching non-experts how to use protein structure and assembly prediction tools and how to benchmark new prediction methods using various datasets assembled in Activity II. Training workflows will be derived and added to the TeSS portal. Activity II will also help coordinate training tasks related to protein structure prediction in the Meet-U initiative. This is an innovative pedagogical initiative created in 2016 that teaches MSc students by involving them in real world research projects, with results evaluated by experts in the field during a final scientific symposium. Meet-U is currently implemented as a collaborative course between three universities of Paris/France area: Sorbonne Université (SU/UPMC), Universités Paris-Sud/Paris-Saclay, and Université Paris-Diderot. It is proposed to implement an international version of Meet-U, involving universities linked to ELIXIR nodes across Europe.\n\nThere has also been some support from the ELIXIR UK node to develop training workflows in protein structure prediction and variant impact analysis. This has enabled pilot work and the establishment of small-scale training workflows in the ELIXIR TESS registry. For all activities, we will organise training workshops for trainers and users, and add workflows to TeSS where appropriate.\n\n3D-Bioinfo community users: The 3D-Bioinfo community bridges several infrastructures and their providers as well as users, namely people and tools of structural biology (Instruct-ERIC, iNEXT), cheminformatics (OpenScreen), system biology (ISBE), molecular simulations (BioExcel) and the proposed community for intrinsically disordered proteins (IDP). In addition, the value of structural data in providing insights into the impacts of genetic variations has led to involvement of some of the 3D-BioInfo participating groups with the ELIXIR Rare Disease Community. Similarly, work in all Activities on structural impacts of residue mutations and other research fields of protein engineering and nucleic acid analogues would clearly enable links with the newly emerging ELIXIR Community on Synthetic Biology. The action plan of this community includes the development of new strains with designed metabolic pathways and expression systems. The synergy of the expertise in the 3D-Bioinfo and Synthetic Biology communities will therefore be very important to implement the new biotech applications that can be expected to be generated through the acquired knowledge and expertise generated by the 3D-Bioinfo ELIXIR community.\n\nThe tools and services offered by the proposed 3D-Bioinfo Community already have many millions of users per year and the new Activities, described above, will undoubtedly broaden this scope.\n\nAs regards potential overlap with Instruct-ERIC, the 3D-Bioinfo community develops tools that go beyond the scope of structural biology and are not covered by the Instruct-ERIC initiatives and efforts. We will work closely with Instruct-ERIC on common areas of interest (e.g. methods for validating experimental protein and nucleic acid structures) and seek support for joint implementations studies.\n\nIndustry and SME involvement – As mentioned already, Activity III (linked to structure-based drug design) is of particular relevance for the pharmaceutical and biotechnology industries, particularly those using computer-aided structure-based drug design programs. Companies involved in the development of scientific software as well as novel bioactive compounds acknowledge the importance of developing benchmarking protocols, and participate in the D3R Grand Challenges by providing undisclosed data. Furthermore, as regards Activity I, PDBe-KB has interactions with OpenTargets platform and provides annotations to major data resources (UniProt, InterPro and Pfam). Development of data standards and distributable infrastructure would further improve accessibility of the added value annotation data for industry users where all four Activities will contribute. We also expect to build up links to the pharmaceutical Industry via the collaborations with BioExcel, and Instruct-ERIC. Future 3D-Bioinfo Activities around protein and enzyme engineering are expected to provide strong links with the industry and it is anticipated that these links will very much strengthen the competitiveness of the developing European biotechnology SMEs.\n\nClearly the ontologies and data exchange formats established and endorsed through 3D-Bioinfo enabled collaborations will be valuable in a global context as well as across Europe. In fact many of the initiatives we will foster such as PDBe-KB, CAPRI and CAMEO already involve other international partners outside Europe. Furthermore, 3D-Bioinfo groups are involved in organising the international community-wide CASP benchmarking of protein structure prediction. We will support and where possible engage in initiatives for global health, for example the Global Alliance for Genomics and Health (GA4GH). We plan to present activities and output from 3D-Bioinfo in special sessions or technology tracks at the European ECCB and international ISMB conferences, the latter of which is held in Europe on alternate years. This will publicise our activities to the wider bioinformatics community, enable us to recruit additional European participants and promote links with other international initiatives.\n\n\nConclusions\n\nThis proposal capitalises on the extensive European structural bioinformatics expertise. It provides a discussion and action framework for joint development of current and future activities within the European structural bioinformatics community. 3D-Bioinfo will very much foster the interactions with experimental research groups to efficiently reach a better understanding of proteins and their functional properties at a quantitative level. 3D-Bioinfo will also foster focused outreach and training activities. The outlined aims of our planned Activities should facilitate valuable coordination for optimal use of resources and exploit ELIXIRs platforms to good purpose. As demonstrated in our background to this paper and the many tools/resources listed in the BioTools, Europe is very strong in this area of research and the establishment of the 3D-Bioinfo Community will assist in promoting research interactions, integration of data and tools and standardised practices, making it even stronger. Furthermore, the Community will facilitate the translation of structurally derived insights - in medicine (pharmaceuticals, diagnostics), agriculture, and sustainable production methods. Finally, 3D-Bioinfo will facilitate collaborations worldwide and promote European research in structural bioinformatics, on a global scale.\n\n\nData availability\n\nNo data are associated with this article.",
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}
|
[
{
"id": "63104",
"date": "21 May 2020",
"name": "Sjoerd Jacob De Vries",
"expertise": [
"Reviewer Expertise Computational molecular modeling",
"structural bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary The authors describe the main goals and provide a road-map of the 3D-Bioinfo community, a new Elixir community. A first part states the major contributions and the leading role of many European teams and initiatives in the structural bioinformatics field. Then, the current limitations and challenges faced by this field are discussed, as well as current European initiatives to address them. The core of the paper is the detailed presentation of the goals of the 3D-Bioinfo community, subdivided in activities (or topics) and few main tasks per activity. Details on the implementation of those tasks and on their interconnections are also provided, as well as their complementarity with other European initiatives (especially within ELIXIR).\nMajor comment\nIn the paragraph “Develop a knowledge portal to user-friendly bioinformatics and computational tools for modeling conformational flexibility of proteins”, it is surprising to see no mention of any task to define common standards and formats in the description of proteins flexibility.\nMinor comments\n“However, many challenges still exist. It remains computationally expensive to build 3D models on a proteome-wide scale” => Yet this aim is being addressed by the SWISS-MODEL initiative. This assertion should be justified.\n“Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design.” => As I understand it, those educational and training efforts are targeting non-informatician structural biologists, and the idea would be to provide them enough insights in 3D-bioinfo for them to foresee possible applications on their system, and apply tools or seek collaboration in 3D-bioinfo. If the training efforts target young 3D-bioinformaticians, then the link with facilitating applications is unclear. The audience could be mentioned here, for clarity.\n“Structural bioinformatics tools link sequence and structure data to predict protein functional sites” => This sounds like a (restrictive) definition of Structural bioinformatics tools. “[Some] structural bioinformatics tools “ would be more correct.\n“As for protein structure prediction, integration of data on sites predicted by different methods will increase both coverage and accuracy.” => This assumption could be softened , as the success of this integration is expected but not certain.\n“This enables the design of new experiments to study the function of macromolecules as well as rational design of proteins[,RNA ?] and drugs, to modify their function and properties.”\n“European structure-based tools have facilitated enzyme reaction mechanism studies by chemists and biochemists.“ => some references would be welcome here\n“The first workshop of the Activity IV is going to take place in May 2020” => This should be updated\n“The above mentioned themes will not only form the initial focus for 3D-Bioinfo but the steering committee will actively monitor the emergence of new technologies and/or new research fields relevant for bioinformatics approaches, which then can be fostered further as new activities. “ => why “will not only form the initial focus”, if the other focuses are supposed to emerge in the future?\nCosmetic comments\n[ ] = to add ; { } = to remove\nThese are small typo or missing comas that are pretty harmless individually, but some can be misleading. And summed up, they impair a fluid reading of this already quite dense paper.\n“The technological developments in MX in the previous decade, largely catalysed by the structural genomics initiatives[,] and the on-going revolution in the field of cryo-EM”\n“some of the most critical contributions to building protein 3D models from structural templates of homologous proteins{,} happened in Europe in the 1990s”\n“Importantly[,] European groups have made major contributions to initiatives “\n“In particular, the RNA-Puzzles experiment for evaluation of RNA structure prediction methods, and a series of associated workshops[,] have been introduced in Europe,”\n“(i.e. CASP27, CAMEO28 and CAPRI29,30 [ ) ] for assessment of the prediction “\n“Below we detail{,} the specific thematic aims of each Activity”\n“We will bring together the community experts to define data standards for different types of annotations [,] and integration of these annotations using a community-driven data exchange format will facilitate finding,”\n“Extend the content of OpenEBench by adding {a} knowledge base integrating software tools for modeling the 3D struc-ture of proteins, protein complexes and assemblies.”\n“methods for generating[,?] scoring, and ranking models of protein complexes”\n“Benchmarking studies performed so far [-] including those carried out in the CSAR52 or D3R Grand Challenge53 (...) - rely on datasets of limited size and diversity (…).”\n“Developing benchmarks dataset[s] / Developing [a] benchmarks dataset”\n“In order to foster new developments[,] focused courses/workshops will be organised on such topics”\n“ exploit ELIXIR{s} platforms “\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": "62621",
"date": "22 May 2020",
"name": "Silvio C.E Tosatto",
"expertise": [
"Reviewer Expertise bioinformatics",
"computational biology",
"structural bioinformatics",
"intrinsically disordered proteins",
"databases",
"tools",
"infrastructure"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Orengo et al. describes the proposed activities of the ELIXIR 3D-Bioinfo user community. This is a useful and necessary contribution to describe the scope and plans of a new ELIXIR user community. The introduction provides a recapitulation of the uses and relevance of structural bioinformatics in different contexts. As the authors note, this is a mature field where several key data resources and initiatives alredy exist. The manuscript reads well and tries to make convincing arguments for the proposed activities.\nSpecific comments:\nFrom the scientific point of view:\nLimits of structural data. Structural data is very important for many purposes, and the authors make this point clearly. However, it has also limits which should be spelled out clearly to avoid the impression of being a panacea. One limit is the availability of experimental data, with some molecules defying structural characterization. A more specific limit is also related to intrinsically disordered proteins (IDPs). This is a different (sub-)field which is not covered by 3D-Bioinfo and has spawned a separate ELIXIR user community with different priorities dictated by the lack of structural data.\n\nConformational variability. This important aspect is mentioned several times and forms a continuum between fully rigid proteins on one end and IDPs at the other. It is important to clarify this concept in the manuscript. By doing so it also offers areas for further collaboration in ELIXIR across user communities.\n\nProteomics Standards Initiative (PSI). Part of the Human Proteomics Organization (HUPO), it should be referred to as HUPO-PSI. This is the main standardization body in the field and an obvious choice for promoting the standards 3D-Bioinfo aims to establish. As such, it should be introduced in the manuscript and its implications discussed. Of note, at present there is no HUPO-PSI working group in the areas covered by 3D-Bioinfo.\n\nActivity 1. The PDBe-KB part is well described and convincing. There is clear potential for synergies on representing IDP data.\n\nActivity 2. The dscription appears somewhat repetitive and its focus may be improved. OpenEBench is a good idea for CAMEO and CARPI, but how can integration be optimally achieved? The comformational modeling portal part is also a bit unclear, especially in light of the overlap with IDPs. A suggestion would be to highlight this as a way to foster collaboration with the IDP user community.\n\nActivity 3. The relevance of protein-ligand interactions is out of question. Since this field is of such pharmaceutical relevance, it is also quite mature. It is however not immediately clear what novel benefit 3D-Bioinfo can provide in terms of instrastructure beyond coordination.\n\nActivity 4. This is scientifically perhaps the most \"novel\" part of the proposed activities, as nucleic acid structures have traditionally been a minority in the field. The description seems more typical of a COST Action setup though and the link to ELIXIR should be improved. E.g. how can the ELIXIR services help to achieve the goals of this activity?\n\nFuture 3D-Bioinfo activities. This section appears somewhat redundant. In the Green Chemistry example it is unclear how this relates to ELIXIR.\n\nISCB. Given the prominent role of at least one main author in the ISCB leadership, this reviewer was expecting a stronger commitment regarding synergies with the society and its COSIs (most notably 3D-SIG). The goals clearly overlap and can benefit from each other.\nFrom the ELIXIR point of view:\nPlatforms. The description of interactions with ELIXIR platforms appears to denote a somewhat cursory knowledge of their activities. The interaction with OpenEBench is well described and convincing. The Data platform description is good for PBDe-KB but after this does not appear related to platform activities. The Compute platform description is also unclear. In all cases, the question is: how will 3D-Bioinfo make convincing use of platform services?\n\nOther user communities. A more thorough description of synergies with existing ELIXIR user communites would be welcome. This should also be separated from non-ELIXIR initiatives (e.g. Instruct, iNEXT, etc.). Of note, the IDP user community is already established and not proposed. Likewise, \"Synthetic Biology\" should probably be Microbial Biotechnology. The Proteomics community is an easy connection but missing. Other communities, e.g. Plants, may also be relevant. In general, it would be good to have specific items for collaboration listed.\nMinor points:\nThe manuscript is somewhat redundant and a bit lengthy at 16 pages without references. Streamlining it would improve focus and can contribute to mitigate some of the concerns above.\n\nThe text refers extensively to \"Europe\" and \"European\" resources, ca. 60 times throughout. While this is a truism for ELIXIR, it sometimes feels just a bit too much emphasis in its present form.\n\nFigure 1 contains data for the PDB up to the year 2016 (a), 2017 (b) and 2018 (c). It should be updated and unified with data until 2019.\n\nWhen listing COST Actions (p. 8, left column, bottom), a unified format should be used. I.e. either list all titles or none.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "63141",
"date": "09 Jun 2020",
"name": "Roland L. Dunbrack",
"expertise": [
"Reviewer Expertise Structural bioinformatics and protein structure prediction"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion article summarizes the activities, both existing and planned, of the 3D-Bioinfo Community within the ELIXIR biological data infrastructure project in Europe. As such, it represents a useful statement of purpose that can be used to guide the efforts of the many participating groups and in the future as a potential means of evaluating the success of those efforts.\n\nThe first section of this article (“Major European contributions in structural bioinformatics”) describes the historical impact of European research groups in the field of structural bioinformatics. These contributions are extensive and indisputable but I am not sure that most scientists will find it very interesting. I suppose it is necessary for European funding bodies to hear it, and in the United States we often find ourselves doing the same thing while trying to secure funding for bioinformatics infrastructure like the PDB from the National Institutes of Health and the National Science Foundation. Perhaps this section could be shortened, or it could briefly mention parallel developments in other parts of the world. For instance, Helen Berman’s efforts in transforming and remediating the PDB into a modern bioinformatics infrastructure resource at Rutgers University starting in 1998 and the establishment of the wwPDB in 2003 with PDBe and PDBj deserve some comment.\n\nThe second section briefly describes “Major challenges that 3D-Bioinfo will help to address” including proteome-wide protein structure prediction, establishing benchmarks and confidence assessments for structure prediction accuracy, evaluating the impact of sequence variants on protein function and disease, improvements in structure determination by cryo-EM, SAXS, integrative modeling, and other methods that depend on the appropriate use of high-resolution structural data from crystallography, and the prediction of protein-ligand interactions. All of these require significant infrastructure and integration and interoperability of tools and data, and 3D-Bioinfo is well placed to operate in this sphere. All of these make sense, even if they are fairly obvious.\n\nIt may be an unpopular opinion, but I don’t think protein structure prediction on the proteome scale is such a worthwhile goal, except for organisms that are of significant experimental interest. Because we can make models of millions of proteins from 100s or 1000s of organisms, doesn’t mean we should. Most of that data would be unused. The fact that it “remains computationally expensive to build 3D models on a proteome-wide scale” as the article states is not really an issue. The more important issue is making biologically relevant models of proteins – as homo- and heterooligomeric complexes, interacting with ligands and nucleic acids, and in different functional states (e.g., the active and several inactive conformations of kinases).\n\nOne challenge that was left out is the validation of experimental structures at the atom/residue level (amino acids, nucleic acid bases, and ligands), and more importantly, their re-refinement with new methods as they are developed. I am a fan of two tools developed by some of the authors on this paper, EDIA for evaluating electron density in X-ray crystal structures on an atom-by-atom basis, and the PDB-REDO database of re-refined crystal structures.\n\nThe next section is a general statement of the goals of 3D-Bioinfo: 1) improved interoperable data standards 2) planning for sustainability and integration of data resources; 3) standards, benchmarking, and validation of prediction tools; 4) access to high compute facilities; 5) integration of structural data with other biological data and databases; and 6) training and outreach in bioinformatics. These are all very worthy, if rather broad and general goals.\n\nThe next section on “Links between 3D-Bioinfo and the wider European research environment” reads more like a grant progress report and is probably of little interest to the general reader, especially the section on the launch meeting, which could be deleted. This section mentions collaboration with the Protein Structure Initiative (PSI) by some of the current 3D-Bioinfo participants, but the PSI was disbanded in 2015 and this reference should be removed. This section mentions Instruct-ERIC, and it would be helpful to describe what are the similarities and differences in the goals of 3D-Bioinfo and Instruct-ERIC, which may not be that familiar to non-European structural biologists. Instruct-ERIC is more focused on experimental technologies.\n\nFinally, we get to the meat of the matter – the four “Activity” areas: I. Infrastructure for FAIR structural and functional annotations; II. Open resources for sharing, integrating and benchmarking software tools for modelling the proteome in 3D; III. Protein-ligand interactions; IV: Tools to describe, analyze, annotate, and predict nucleic acid structures. (Full disclosure: I am a participant in Activity II).\n\nThe first activity (I) is to improve annotations of protein structures, in particular to bring 3rd party annotation resources into the PDBe-KB (knowledge-base) via a standard data exchange format (e.g., json-based formats). These will include functional site annotations, predicted structures, and impact of disease-associated variants on structure. This is appealing to me, since my group generates these kinds of annotations, for example on antibodies, kinases, and protein-protein interactions. Access to these annotations via the PDB would greatly increase the impact of such resources and the depth of annotation of PDB structures. One example that is already in practice is the import of QSBIO and 3Dcomplex annotations for protein assemblies of X-ray structures in the PDB.\n\nActivity II is focused on integrating and benchmarking software for modeling proteins in 3D, including in particular modeling or annotating protein assemblies and conformational flexibility. This aspect of 3D-Bioinfo will include integration of CAMEO and CAPRI, both of which assess structure predictions of proteins or protein complexes, into OpenEBench, which is also part of ELIXIR. It will also involve developing benchmarks for the prediction of the structures of protein assemblies (e.g. for benchmarking docking calculations) and providing a portal for community-derived benchmarks. While there are many such benchmarks, they are not fully integrated into one resource which will likely be productive in the way that CAMEO, CAPRI, and CASP have been. This section could use a more succinct statement of what is missing in existing benchmarks and what 3D-Bioinfo would do to fill the gaps.\n\nActivity III is focused on developing and disseminating benchmarks for modeling protein-ligand complexes, including peptides and macrocyclic compounds, and their affinities. The inclusion of experimentally determined non-active compounds (for the construction of negative data sets) is an important suggestion in the paper. Another goal is to “open the world of structure-based modeling to the broader science community.” Worthy, but easier said than done, and it is not clear what this would look like exactly.\n\nActivity IV is focused on refinement of experimental structures, structural bioinformatics, structure prediction, and benchmarking studies of nucleic acid structures, which, as the authors point out, lag behind comparable efforts on proteins. As with the other Activities, the efforts will be on establishing benchmarks, collecting software tools, and enhancing interoperability. The authors explicitly propose bringing additional research groups outside of 3D-Bioinfo into Activity IV, presumably including those outside of Europe, which would greatly strengthen all of the activities of 3D-Bioinfo.\n\nThe paper concludes with a discussion of further interactions of 3D-Bioinfo with other research communities including: (1) ELIXIR “platforms” on Interoperability, Data, Tools, Compute, and Training; (2) European bioinformatics infrastructure initiatives such as those on cheminformatics, systems biology, and intrinsically disordered proteins; (3) connections with industry; and (4) global initiatives. The last of these is given rather short shrift, considering that the efforts of a European 3D-Bioinfo project will not succeed if they don’t include input and adoption by research groups worldwide. In particular, it is surprising not to see any discussion of interactions with the RCSB or PDBj, which are after all components of the World-Wide PDB (wwPDB), along with PDBe.\nVery minor:\nFigure 2: typo, “Uniport” --> “Uniprot”\n\np. 14: “EMBO courses on Integrative of Biomolecular Complexes” --> “EMBO courses on Integrative Modeling of Biomolecular Complexes”\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": "63105",
"date": "11 Jun 2020",
"name": "Andras Fiser",
"expertise": [
"Reviewer Expertise computational structural 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 manuscript of Orengo et al presents the goals of the Elixir initiative in the context of historical achievements in the field of 3D Bioinformatics. It is a conceptual piece to provide guidance to an entire field and therefore I would reflect on that.\n\n‘Computational approaches in biology’ always suffered from an identity problem and I believe this manuscript does not help to resolve it and as such it might be a missed opportunity. One major issue is if one approaches computational structural biology as a discipline or a technology. I believe Elixir efforts all about boosting the potential of this discipline but the scope of this effort is still presented rather as a technology that can support other disciplines.\n\nThe first sentence of the abstract immediately leaves this question un-answered “Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community.”. By leaving out aspects of “modeling” from the possible contribution, which I believe is a frequent and important aspect of computational structural biology, the authors restrict the activities for retrospective analysis of experimental data, to “bioinformatics”, i.e to “organize, analyze an manipulate data”. And do not make the argument that “modeling”, i.e. gaining new, testable hypothesis and generating new knowledge is also part of this field (e.g. structural modeling of proteins without experimental structures; docking receptors and ligands in so far unseen complexes; predicting new receptor ligand interactions; predicting and modeling protein drug interactions; modeling /simulating protein motions; modeling enzyme kinetics with molecular dynamics or quantum mechanical simulations; etc.).\n\nNIH made an attempt to distinguish between “bioinformatics” and “computational biology”. If I try paraphrase it in a 3D context, 3D-Bioinfirmatics is: research, development, or application of computational tools and approaches for expanding the use of 3D biological data, including those to acquire, store, organize, archive, analyze, or visualize such data. In contrast to 3D-Computational Biology of biomolecular structures, which is the development and application of data-analytical and theoretical methods, mathematical modeling and computational simulation techniques to the study of macromolecular structures.\n\nThis review so far was concerned with the first sentence of the Abstract only. The rest of the paper does touch on a number of innovative, activities that generate new knowledge, i.e. a discipline like activity, but at least to this reviewer this is not formulated in the framework enough.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-278
|
https://f1000research.com/articles/9-190/v2
|
22 Apr 20
|
{
"type": "Research Article",
"title": "Identification Sus scrofa and Mus musculus as potential hosts of SARS-CoV-2 via phylogenetic and homologous recombination analysis",
"authors": [
"Xiaopeng Hu",
"Weixin Li",
"Zhendan He",
"Fengxue Zhang",
"Xiaopeng Hu",
"Weixin Li",
"Zhendan He"
],
"abstract": "Background: Previously, most studies focus on the wild animal being sold in the Wuhan Huanan seafood wholesale market, neglecting that the livestock living around other place could also be the original hosts. Methods: First, relative synonymous codon usage was utilized to analyze the potential hosts of SARS-CoV-2; Then cluster SARS-CoV-2 and related coronavirus through the phylogenetic tree. Next, we used Recombination Detection Program to identify the possible recombination region, as well as verifying via Simplot. Results: Related coronavirus from porcine or murine sources may faciliatate the evolution and reorganization of SARS-CoV-2. Conclusions: Overall, to our knowledge, this is the first paper to illustrate that swine and mice could be probable reservoirs for the SARS-CoV-2.",
"keywords": [
"SARS-CoV-2",
"Sus scrofa",
"Mus musculus",
"phylogenetic analysis",
"homologous recombination analysis"
],
"content": "Introduction\n\nA novel coronavirus, SARS-CoV-2, was recently reported in the city of Wuhan, Hubei province, China, causing severe respiratory diseases as well as epidemic all around the China. The inflection point of confirmed cases didn’t occur until February 2020, with the first patient hospitalized on the 12th of December 20191. On 29th February, 573 new confirmed cases of novel coronavirus infection were reported on the Chinese mainland, bringing the total to 78,630. There were 35 new fatalities reported daily, with the cumulative fatalities were up to 2761. Meanwhile, outside the Chinese mainland, more than 5000 cases have been confirmed in Asia, in places such as Japan, Singapore, Thailand and South Korea, in Europe, in places like Germany and France, and in the Americas. Consensus that the SARS-CoV-2 originated from bats has been reached2. However, intermediate hosts are deemed as having mediated human infection via gradually adapting the mechanism of transcription and translation in the human body.\n\nThe exact putative parent of SARS-CoV-2 remains uncertain. Frequent contact between humans and swine could lead to a higher risk of cross-species transmission or virus recombination. For the sake of identifying intermediate host, relative synonymous codon usage (RSCU) analysis was applied to evaluate the potential diversity of species acting as reservoirs. Phylogenetic and homologous recombination analysis were also used to illuminate the correlated coronavirus hosting in the most possible host.\n\n\nMethods\n\nPorcine and murine coronavirus were refined and downloaded from NCBI virus database (Table S1, Extended data B)3. Here, the mitochondrial genes represent the whole genome of potential host (Table S2, Extended data B)3. ClustalX 1.83 was applied to align the sequences.\n\nMEGA X (v1.0.3) was used to construct the phylogenetic trees using the Neighbor-Joining method4 (The input GenBank accessions are shown in Table S1, Extended data B)3. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches5. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All genome sequences were aligned before implementing of phylogenetic analysis.\n\nIn order to identify the relative synonymous codon usage (RSCU) bias of the SARS-CoV-2 and its potential host, the coding sequences of suspect species were calculated using CodonW 1.4.2. (Table S4, Extended data C)6. Whole coding sequences of the genomes, downloaded from NCBI, were utilized for identifying the host among different viruses7 (the input GenBank accessions are shown in Table S1, Extended data B)3. The RSCU were calculated with CodonW 1.4.2. The heat map clustering of RSCU was realized via applying MeV 4.9.0. The homologous analysis was executed via analyzing the pairwise distance of mitochondrial genes from the potential host animal. The pairwise distances were computed with MEGA X via the bootstrap test (1000 replicates) to evaluate potential hosts8.\n\nThe genome sequences of bat RaTG13 (MN996532.1), murine JHM (AC_000192.1), Human HKU1 (NC_006577.2), PEDV H11-SD2017 (MH708243.1) and PEDV YN15 (KT021228.1) were obtained from the NCBI Virus database. Potential recombination events of SARS-CoV-2 were first implemented with Recombination Detection Program v4 (RDP4), then Simplot (version 3.5.1) was utilized to verify the possible breakpoints. The BootScan method were taken into the RDP4 analysis. Potential recombination events were characterized with similarity plots, with possible regions of recombination indicated.\n\n\nResults\n\nRSCU has been widely utilized to analyze the association between virus and potential host7. The output from RSCU heat map, based on the Euclidean distance, inferred that both Sus scrofa and Mus musculus have similar synonymous codon usage bias with SARS-CoV-2 (Figure 1A). The Euclidean distance between Sus scrofa and SARS-CoV-2 is minimal, hinting that SARS-CoV-2 could effectively use porcine translation machinery better than that of other animals, suggesting that the epidemic SARS-CoV-2 might originate from swine. The pairwise distance between the SARS-CoV-2 complete genome and the potential host mitochondrial genome also supports the above judgments. Regarding the pairwise distances of SARS-CoV-2, both swine and mice presented shorter than snakes, marmots, mink, bats and humans and (Figure 1B). Pairwise distance between the SARS-CoV-2 and Sus scrofa is 14.89, and for SARS-CoV-2 and Mus musculus is 14.94. This result indicates that SARS-CoV-2 may originate from both swine and mice.\n\n(A) Heat map of relative synonymous codon usage (RSCU) derived from the complete genome of SARS-CoV-2, as well as the mitochondrial genome of diverse animals (Sus scrofa, Mus musculus, Najaatra, Mustela Pulourius, Marmota flaviventris, Rhinolophus sinicus, Homo sapiens). (B) Homology analysis between the SARS-CoV-2 and different animal species (Table S4), Pairwise Distance was applied to evaluate the homology as compared with SARS-CoV-2 via MEGA-X.\n\nThe 21 PEDV strains were filtered from 2010~2020 in Hubei based on the NCBI virus database (The GenBank accessions were shown in Figure 2). Phylogenetic analysis of the coronavirus (HKU15, PHEV, NL63, H11-SD2017, 229E, HKU2, TGEV, etc.) derived from varied species (bats, swine, humans), representing the sister lineage to SARS-CoV-2 with 99% bootstrap support (Figure 2). RSCU revealed that related coronaviruses have similar synonymous codon usage bias with SARS-CoV-2 (Figure 3). The other PEDV strains obtained from Hubei also showed the closely phylogenetic correlation with SARS-CoV-2. Overall, the close phylogenetic relationship to Sus scrofa provides evidence for bat-swine axis, being one of the origins of SARS-CoV-2.\n\nThe neighbor-joining tree (bootstrap n=1,000; p-distance) was exerted to represent the evolutionary history of the taxa analyzed. Phylogeny-based geographical dissection of 21 PEDV strains (Brown box) derived from Hubei. Information about the 21 PEDV strains is shown in Table S1 Extended data B3.\n\nThe heat map of RSCU derived from the complete genome of SARS-CoV-2 and other polyphyletic coronaviruses. The Euclidean distance was calculated to cluster the related coronavirus.\n\nMeanwhile, the result of RSCU also presented that both SARS-CoV-2 and related coronavirus lean towards to having similar synonymous codon usage bias (Figure 3 and Table S5, Extended data C)6. Therefore, the origination of SARS-CoV-2 could be further focus on the coronavirus isolating from swine and mice. Particularly, the relationship with PEDV H11-SD2017 and PEDV YN15 needed to be further studied.\n\nTo our knowledge, this is the first study to report that porcine and murine coronavirus may attend the reorganization of SARS-CoV-2. The RDP4 estimated the possible reorganization regions for SARS-CoV-2 (Table 1, Figure 4A). Furthermore, SimPlot analysis confirmed the homologous recombination of sequence similarity between SARS-CoV-2 and coronavirus from potential hosts. The potential recombination breakpoints (16205-16358nt) are shown in red dashed lines (Figure 4B), indicating recombination between PEDV YN15 and the murine hepatitis virus JHM when SARS-CoV-2 was queried. In addition, the region between 20923 and 21181 nt also indicated a recombination event taking place between PEDV H11-SD2017 and Human coronavirus HKU1 when SARS-CoV-2 was queried (Figure 4C).\n\nMN908947.3: Wuhan_seafood_market_pneumonia_virus_isolate_Wuhan-Hu-1_complete_genome; KT021228.1: Porcine_epidemic_diarrhea_virus_strain_YN15_complete_genome; AC_000192.1: Murine_hepatitis_virus_strain_JHM_complete_genome; NC_006577.2: Human_coronavirus_HKU1_complete_genome; MH708243.1: Porcine_epidemic_diarrhea_virus_strain_H11-SD2017_complete_genome.\n\nThe similarities to different reference sequences are indicated by different colors shown in the legend box at the top. (A) The sequence of SARS-CoV-2(query), Bat coronavirus RaTG13, Human coronavirus HKU1, Murine hepatitis virus JHM, PEDV YN15 and PEDV H11-SD2017 were assessed with similarity plot. (B) The enlarged figure identifies the homologous recombination region (16205–16358 nt) of PEDV YN15 and Murine hepatitis strain JHM. (C) The enlarged figure identifies the homologous recombination region(20923–21181nt) of PEDV H11-SD2017 and Human coronavirus HKU1.\n\n\nDiscussion\n\nIn China, the cumulative number of patients diagnosed as infected with SARS-CoV-2 is thought to over 100,000, with more than 2000 deaths. It is the most severe public health emergency since the outbreak of SARS 17 years ago9. Local residents were reined with anxiety and confused during the virus outbreak, although the Chinese government has taken substantial action to prevent and control the spread of the virus. However, multiple different messages could disrupt the attention of medical workers and science researchers. Current research about the origin of SARS-CoV-2 mostly focuses on wildlife, since the first case was thought to be highly associated with wild animals in Wuhan sea food market.\n\nThe origin of SARS-CoV-2 had caused great concern to the public. Natural variation was the dominant view holding by most scholars, believing that bats and other wild animals provide reservoirs for the virus. However, coronavirus from bats is unlikely to infect humans directly; one or two intermediate hosts may facilitate the homologous recombination, enabling the coronavirus to gradually adjust to the human genetic code, then survival and breeding successfully.\n\nFrom our perspective, natural variation would be a more reasonable explanation for the origin of SARS-CoV-2. However, the wild animals in Wuhan seafood market should not accept all the liability, since there is evidence that patients with early infection didn’t have any contact with the market9. Some researchers10 have also pointed out that the Wuhan seafood market is not the only area of origin, indicating that another creature may act as intermediate host apart from those animals being sold at the market. In our study, the captivity animal, Sus scrofa (swine) and Mus musculus (mice) were suspected to be critical hosts of SARS-CoV-2.\n\nOur finding supports the theory of natural variation. Natural variation believes that people get infected because they eat or come into contact with intermediate hosts. SARS-CoV-2 was found to be 96% identical at the whole-genome level to the bat coronavirus2. Other reports state that snakes, mink, and pangolins could be potential hosts for SARS-CoV-211.\n\nEither wild animals or reared livestock could serve as hosts. It has been reported that Wuhan seafood market may not be the only source of novel virus spreading globally because the earliest patient became ill on 1 December 2019 and had no epidemiological link to the seafood market or later cases. The official details about the first 41 hospitalized patients showed 13 of the 41 patients had no link to the marketplace at all9. One possible hypothesis is that the cross-species transmission has occurred in other places before the outbreak of Wuhan Huanan Seafood market.\n\nPrevious study into fatal swine acute diarrhea syndrome (SADS) revealed that SADS-related coronavirus was responsible for a large-scale outbreak of fatal disease in pigs in China12. Here we discovered Porcine epidemic diarrhea virus (PEDV) periodicity burst in China13. Among one of the strains, H11-SD2017 showed closely affiliation with SARS-CoV-2 via implementing relative synonymous codon usage (RSCU) and phylogenetic analysis. Swine-to-human cross species transmission may explain why many patients with coronavirus disease-19 (COVID-19) not only suffer from severe respiratory diseases, but also diarrhea9.\n\nIn the past 10 years, PEDV has spread into most provinces with the swine industry in China. Hubei was the first region to be infected with PEDV strain CH/HBQX/10 in 2010; from then on, more and more provinces reported the presence of PEDV13. The spread of PEDV has gone beyond its initial geographical limitation. There were more than 300 PEDV strains clustered into pandemic, meaning the significant natural variation took place in the spread of PEDV in different regions. Further analysis among 21 strains of PEDV and coronavirus from varied species indicate that swine and mice could be other hosts of SARS-CoV-2 (Figure 1A, Figure 2 and Figure 3).\n\nRDP4 and Simplot analysis helped us better understand the homologous recombination of SARS-CoV-2 (Table 1, Figure 4). It verified that not only porcine coronavirus, but also murine coronavirus, experienced recombination events. Therefore, we speculate that SARS-CoV-2 may originate from the bat firstly, undergoing a series of recombination events, with swine and mice playing critical role in mediating cross species transmission.\n\nPairwise analysis of distance also indicated that Mus musculus could be a possible host of SARS-CoV-2 (Figure 1B). Previously, the Chinese Centers for Disease Control said that 33 of the samples were positive for the novel coronavirus nucleic acid. The positive samples were distributed among 22 stalls and a garbage truck in the Wuhan Huanan seafood market, so the outbreak is highly suspected to be related to the wildlife trade. But how could wild animal movement around the market lead to cross-infection with disparate species in different regions? One plausible phenomenon is that mice could be infected with SARS-CoV-2 firstly, transferring the infection to other wild species in the market. Thus, the role of mice in the market deserves more attention. In order to verify the exact intermediate host, mice and swine living around Wuhan should be collected for further proof test basing on Koch’s postulates\n\nEventually, the rear livestock should be deserved more notice apart from wild vertebrate creatures. To our knowledge, this is the first study to illustrate that swine and mice are the probable livestock reservoir for the SARS-CoV-2. Furthermore, the mice around the seafood market may also be involved in the cross transmission of the virus to some extent. All of those output based on the bioinformatics analysis, further identification of host should be verified from isolation and other experiments.\n\n\nData availability\n\nThe genomic data assessed in this study are described below in the Extended data.\n\nFigshare: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. https://doi.org/10.6084/m9.figshare.1192558814.\n\nThis project contains Extended data A: PEDV distribution in China from 2010–2020. (The 329 PEDV genomes assessed in this study.)\n\nFigshare: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. https://doi.org/10.6084/m9.figshare.119256933.\n\nThis project contains Extended data B: The GenBank accession numbers used for analysis. (Contains accession numbers, strain name and species used for phylogenetic analysis.)\n\nFigshare: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. https://doi.org/10.6084/m9.figshare.119256546.\n\nThis project contains Extended data C: RSCU analysis of diverse genome. (Contains RSCU analysis of codons derived from SARS-CoV-2.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe are appreciated with the sharing of SARS-CoV-2 complete genome (GenBank accession MN908947) from Prof. Yongzhen Zhang research team, as well as the related genome GenBank accessions from other researchers.\n\n\nReferences\n\nWu F, Zhao S, Yu B, et al.: A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798): 265–269. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu X: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11925693.v1\n\nSaitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4): 406–425. PubMed Abstract | Publisher Full Text\n\nFelsenstein J: Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985; 39(4): 783–791. PubMed Abstract | Publisher Full Text\n\nHu X: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11925654.v1\n\nChen LL, Ou HY, Zhang R, et al.: ZCURVE_CoV: a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing SARS-CoV genomes. Biochem Biophys Res Commun. 2003; 307(2): 382–388. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTamura K, Stecher G, Peterson D, et al.: MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12): 2725–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang C, Horby PW, Hayden FG, et al.: A novel coronavirus outbreak of global health concern. Lancet. 2020; 395(10223): 470–473. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu WB, Zhang L, Tang GD, et al.: Decoding evolution and transmissions of novel pneumonia coronavirus using the whole genomic data. [ChinaXiv: 202002.00033], 2020. Reference Source\n\nJi W, Wang W, Zhao X, et al.: Cross-species transmission of the newly identified coronavirus 2019-nCoV. J Med Virol. 2020; 92(4): 433–440. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou P, Fan H, Lan T, et al.: Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature. 2018; 556(7700): 255–258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun M, Ma J, Wang Y, et al.: Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia. J Clin Microbiol. 2015; 53(5): 1484–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu X: Identification Sus scrofaand Mus musculus as potential parasitifersof SARS-CoV-2 via phylogenetic and homologous recombination analysis. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11925588.v2"
}
|
[
{
"id": "290448",
"date": "13 Jun 2024",
"name": "Jonas Wolf",
"expertise": [
"Reviewer Expertise Biostatistics",
"virology",
"bioinformatics",
"biochemistry",
"immunology",
"molecular evolution."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 titled \"Identification Sus scrofa and Mus musculus as potential hosts of SARS-CoV-2 via phylogenetic and homologous recombination analysis\" explores a novel perspective on the origins and potential hosts of the SARS-CoV-2 virus. Traditionally, investigations into the origin of SARS-CoV-2 have centered on wildlife in the Wuhan Huanan seafood market. However, this study shifts focus to domestic animals, specifically Sus scrofa (pigs) and Mus musculus (house mice), providing a fresh and potentially crucial viewpoint on the epidemiological landscape of SARS-CoV-2.\nPositive Aspects Novel Perspective: The study takes a commendably innovative approach by considering livestock as potential hosts of SARS-CoV-2, which broadens the scope of research beyond the commonly suspected wildlife. This helps in potentially identifying new pathways for zoonotic transmission which might have been overlooked.\nMethodological Rigor: The use of relative synonymous codon usage (RSCU) to assess potential hosts demonstrates a thorough and sophisticated understanding of viral evolution. The combination of phylogenetic analysis and recombination detection adds robustness to the findings, providing a comprehensive view of the virus's potential to adapt to different hosts.\nUse of Phylogenetic and Recombination Analysis: By clustering SARS-CoV-2 with related coronaviruses and analyzing recombination events, the study offers valuable insights into the evolutionary pathways of the virus. This approach can reveal not only possible host species but also evolutionary mechanisms that might facilitate cross-species transmission.\nPotential Public Health Impact: Identifying domestic animals as potential reservoirs of SARS-CoV-2 could have significant implications for public health policies. It underscores the need for broader surveillance and biosecurity measures that encompass not only wildlife but also domestic and peridomestic animals.\nPioneering Findings: The conclusion that swine and mice could be probable reservoirs for SARS-CoV-2 is groundbreaking. It opens up new avenues for research and underscores the importance of considering a wider range of species in the study of zoonotic diseases.\nOpportunities for Improvement Scope of Host Analysis: While the study focuses on Sus scrofa and Mus musculus, it might have benefitted from a broader examination of other potential domestic animal hosts. Including additional species could provide a more comprehensive understanding of the potential host range of SARS-CoV-2.\nVerification of Host Susceptibility: The study’s conclusions are primarily based on genetic and phylogenetic analyses. Empirical validation through laboratory infection studies or epidemiological evidence would strengthen the claim that these animals can indeed act as reservoirs for the virus.\nDetailed Analysis of Recombination Events: The paper mentions the identification of recombination regions but lacks an in-depth discussion of these events and their potential implications. A more detailed analysis of how these recombination events might influence viral transmission and pathogenicity in different hosts would add significant value.\nContextualizing Findings within Existing Literature: While the study introduces a new perspective, it could benefit from a more thorough comparison with existing research on SARS-CoV-2 hosts. This would help in situating the findings within the broader context of current scientific understanding and highlight how this study advances the field.\nExploration of Ecological and Environmental Factors: The study could also delve deeper into the ecological and environmental factors that might facilitate the interaction between these domestic animals and humans. Understanding these dynamics is crucial for assessing the real-world risk of zoonotic spillover.\nClearer Explanation of RSCU: While the use of RSCU is a strong point, the paper would benefit from a more detailed explanation of how this method works and why it is particularly suitable for identifying potential viral hosts. This would make the findings more accessible to a broader audience.\nDiscussion of Limitations: The study would benefit from a more explicit discussion of its limitations. For example, it could address the constraints of phylogenetic analysis in predicting host range and the limitations of computational methods in the absence of empirical data.\nConclusion Overall, the article \"Identification Sus scrofa and Mus musculus as potential hosts of SARS-CoV-2 via phylogenetic and homologous recombination analysis\" represents a significant contribution to the field of virology and zoonotic disease research. By expanding the focus to include domestic animals, it opens new avenues for understanding the origins and spread of SARS-CoV-2. However, the study would benefit from broader host analysis, empirical validation, and a deeper contextualization of its findings. Despite these areas for improvement, the paper provides a valuable foundation for future research and underscores the complexity of viral host dynamics.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": []
}
] | 2
|
https://f1000research.com/articles/9-190
|
https://f1000research.com/articles/9-78/v1
|
03 Feb 20
|
{
"type": "Research Article",
"title": "Staphylococcus aureus nasal carriage and microbiome composition among medical students from Colombia: a cross-sectional study",
"authors": [
"Niradiz Reyes",
"Oscar Montes",
"Stephanie Figueroa",
"Raj Tiwari",
"Christopher C. Sollecito",
"Rebecca Emmerich",
"Mykhaylo Usyk",
"Jan Geliebter",
"Robert D. Burk",
"Niradiz Reyes",
"Oscar Montes",
"Stephanie Figueroa",
"Raj Tiwari",
"Christopher C. Sollecito",
"Rebecca Emmerich",
"Mykhaylo Usyk",
"Robert D. Burk"
],
"abstract": "Background: The anterior nares are the main ecological niche for Staphylococcus aureus, an important commensal and opportunistic pathogen. Medical students are frequently colonized by a variety of pathogens. Microbial interactions in the human nose can prevent or favor colonization by pathogens, and individuals colonized by pathogens have increased risk of infection and are the source of transmission to other community members or susceptible individuals. According to recent studies, the microbiome from several anatomic areas of healthy individuals varies across different ethnicities. Although previous studies analyzed the nasal microbiome in association with S. aureus carriage, those studies did not provide information regarding ethnicity of participants. Our aim was to assess S. aureus nasal carriage patterns and prevalence among medical students from Colombia, a country of Hispanic origin, and to investigate possible associations of colonization and nasal microbiome composition (bacterial and fungal) in a subgroup of students with known S. aureus carriage patterns. Methods: Nasal swabs from second-year medical students were used to determine prevalence and patterns of S. aureus nasal carriage. Based on microbiological results, we assigned participants into one of three patterns of S. aureus colonization: persistent, intermittent, and non-carrier. Then, we evaluated the composition of nasal microbial communities (bacterial and fungal) in 5 individuals from each carriage category using 16S rRNA and Internal-Transcribed-Spacer sequencing. Results: Prevalence of S. aureus nasal carriage among medical students was 28%. Carriage of methicillin-resistant strains was 8.4% and of methicillin-sensitive strains was 19.6%. We identified 19.6% persistent carriers, 17.5% intermittent carriers, and 62.9% non-carriers. Conclusions: Analysis of nasal microbiome found that bacterial and fungal diversity was higher in individuals colonized by S. aureus than in non-carriers; however, the difference among the three groups was non-significant. We confirmed that fungi were present within the healthy anterior nares at substantial biomass and richness.",
"keywords": [
"Microbiome",
"mycobiome",
"microbiota",
"Staphylococcus aureus",
"bacterial communities."
],
"content": "Introduction\n\nThe anterior nares are significant because they represent the main ecological niche for Staphylococcus aureus, a bacterium that behaves both as commensal and opportunistic pathogen1. Asymptomatic carriage of S. aureus in healthy individuals has high prevalence, especially in children, young adults, and healthcare workers, including medical students2–4. Due to their frequent contact with the general community and healthcare environment, medical students commonly encounter a variety of pathogens that may colonize them. Individuals colonized by pathogens have increased risk of infection, but also they become a source of transmission to other community members or susceptible patients1,5–7.\n\nMicroorganisms residing in a particular anatomical site engage in complex interactions that prevent or favor colonization by pathogens8. The complete collection of microbes colonizing the anatomical areas of the human body constitute the microbiota, which includes bacteria, archaea, viruses, and fungi, intertwined in a complex network of interactions among them and the host9. The genes and genomes harbored by these microbial communities make up the human microbiome10,11. According to recent studies, the microbiome from several anatomic areas of healthy individuals varies across different ethnicities12–15. Although previous studies have analyzed the nasal microbiome in association with S. aureus carriage, these studies have not provided information on the ethnicity of participants, or have used individuals from different ethnic populations16,17. Differences in microbiome composition linked to ethnic background highlight the need to consider and potentially account for ethnic diversity in microbiome research12. This study sought to determine the nasal prevalence and long-term carrier patterns of S. aureus in second-year medical students with Hispanic background. Additionally, we aimed to explore biodiversity of the nasal microbiome (bacterial and fungal) and possible differential abundance of specific taxa among the three categories of S. aureus long-term carriage.\n\n\nMethods\n\nThe Ethics Review Boards of the University of Cartagena (Approval #280313) and New York Medical College approved this study (Protocol # 12697; IRB ID: 12697). The study was conducted between January and June of 2018 and enrolled second-year medical students from University of Cartagena, Colombia, who had not yet engaged in clinical rotations. To prevent sampling bias, we aimed to enroll the complete population of second-year medical students of our institution. Students were recruited via fliers and lecturer announcements. Those who agreed to participate signed an informed consent and completed a written questionnaire on demographics and medical history. Exclusion criteria were recent infections, allergies and other non-infectious pathologies, smoking habits, antibiotic usage in the previous three months, surgeries and hospitalizations in the previous six months. In total 143 out of 158 second-year medical students completed the study. Thus, we enrolled 90.5% of the total population of second-year medical students in our institution in this study. We followed the STROBE cross sectional reporting guidelines18.\n\nNasal swabs were obtained from both nostrils, inoculated into Stuart transport medium (OXOID, England), transported to the microbiology laboratory and processed within 8–18 hours according to described protocols6. S. aureus was identified based on colony morphology, Gram-stain, catalase-test, tube coagulase-test, and latex agglutination-test. Genomic DNA was obtained from each isolate with Wizard® Genomic DNA Purification Kit (Promega, USA) and S. aureus molecular confirmation and methicillin-resistance were assessed by PCR-amplification using specific primers for nuc and mecA genes, respectively. Detailed protocols for all these methods, including primer sequences, have been previously described6. To determine prevalence of S. aureus nasal carriage, each participant was classified either as carrier or non-carrier based on laboratory results obtained from the first nasal swab survey. To establish S. aureus long-term carriage categories, four additional consecutive nasal swabs were obtained from each participant, in three-week intervals. According to definitions proposed by Kluytmas et al.19, participants that yielded five negative cultures for S. aureus were classified as non-carriers; those yielding one to three positive cultures were classified as intermittent carriers; and those yielding four or five positive cultures were classified as persistent carriers.\n\nThis study aimed to describe the microbiome composition in a small group of second-year medical students with a known S. aureus carriage status. At the end of the study, 15 participants with known S. aureus long-term carriage status (5 non-carriers, 5 intermittent-carriers, and 5 persistent-carriers) were randomly selected from the cohort of 143 participants. The 15 selected participants provided an additional nasal swab that was stored at room temperature in Amies-Transport-Medium with Charcoal (Copan Diagnostics, Inc., Murrieta, CA), and sent to the laboratory of Robert D. Burk at Albert Einstein College of Medicine (AECOM) for microbiome analysis. Sample processing was performed according to protocols described by Usyk et al.20.\n\nThe V4 hypervariable region of 16S rRNA gene was amplified using primers 16SV4_515F (GTGYCAGCMGCCGCGGTA) and 16SV4_806R (GGACTACHVGGGTWTCTAAT), with a unique 12-bp barcode Golay-barcoding21,22. PCR conditions were: initial 5min denaturation at 95°C, followed by 15-cycles of 95°C for 1min, 55°C for 1min, and 68°C for 1min, and final extension for 10min at 68°C. For the fungal component of microbiome, barcoded amplicons were generated covering the ITS gene region using ITS1-30F/ITS1-217R primer pair, as previously described by Usyk et al.20 (ITS1-30F: 5’-GTCCCTGCCCTTTGTACACA-3’ and ITS1-217R: 5’-TTTCGCTGCGTTCTTCATCG-3’). PCR conditions were: 3min initial denaturation at 95°C, followed by 35-cycles of 95°C for 30sec, 55°C for 30sec, and 68°C for 2min, followed by final extension at 68°C for 10min. PCR reagents were obtained from Affymetrix (Affymetrix, Santa Clara, CA). PCR reactions were run in GeneAmp PCR-System 9700 (Applied Biosystems).\n\nPCR products were purified using QIAquick Gel Extraction Kit (QIAGEN) and quantified using QubitTM dsDNA High-Sensitivity Assay kit (Life Technologies). Next-generation sequencing library preparation was performed using KAPA-LTP library preparation kit (KAPA Biosystems, Wilmington, MA). Size integrity of isolated amplicons was validated with 2100 Bioanalyzer (Agilent-Technologies, Santa Clara, CA). High-throughput sequencing of libraries was performed using Illumina HiSeq2500 Sequencing System (Illumina, San Diego, CA) with a 2×250-bp paired-end read kit at the Genomics Core Facility of AECOM.\n\nIllumina reads were pre-processed to remove bases that fell below PHRED quality score of 25 using PRINSEQ23. Processed reads were de-multiplexed using sample specific barcode combinations with Novobarcode V1.00. This can also be performed with deML, a program freely available for use under the GPL license. Paired-end reads were merged using free open source PANDAseq v1.20 with default settings24. OTU-clustering and quality filtering was performed using the Quantitative Insights Into Microbial Ecology (QIIME v1.9) software package25. Removal of sequencing noise and sequence chimeras was done with USEARCH v8.026. Sequences were de-multiplexed and clustered into operational taxonomic units (OTUs) with 97% minimum cluster similarity using UCLUST26. Sequences were assigned using UCLUST with Greengenes 13.8 microbial database27. The resulting BIOM table was rarefied to 29,000 reads/sample and statistical analyses were performed after collapsing OTUs at genus level.\n\nSequence reads were processed using open-reference OTU-picking with QIIME v1.9 against the targeted host-associated fungi ITS database (THF1)28 for the reference-based clustering component. VSEARCH v1.4.029 was used to de-replicate reads, cluster reads into OTUs and remove chimeric sequences. OTU-clustering threshold was set at 99% sequence identity to account for fungal heterogeneity. Sequence de-replication and chimera removal were performed using QIIME quality-control protocol. Representative sequences for each OTU-cluster were chosen based on sequence abundance. BLAST was used to assign taxonomy using the UNITE database30. The default behavior of BLAST in QIIME was changed to minimum of 99% sequence identity for taxonomic assignment. Data were processed in R v3.3.131. QIIME outputs were imported into R using phyloseq package v1.22.332 and further processed with vegan v2.5-333, coin v134, and reshape235. Data visualization was performed using ggplot236.\n\nStatistical analyses were performed to assess differences in OTU distribution and abundance between samples and groups. Microbial diversity for bacterial and fungal communities was evaluated within samples (α-diversity) or between samples (β-diversity) using QIIME. Rarefaction to subsampling depth of 29,000 reads/sample or 9,000 reads/sample, for bacteria or fungi respectively, and 5 iterations were performed on all samples to standardize the sequencing effort. Alpha-diversity was measured with Chao1 (richness) and Shannon entropy (OTU-based diversity) index. Beta-diversity was calculated using Bray-Curtis dissimilarity coefficient. To test for dissimilarities in the microbial composition between S. aureus carrier groups, non-metric multidimensional scaling (NMDS) was performed with Bray-Curtis dissimilarity.\n\n\nResults\n\nThe first nasal swab isolated S. aureus from 40 out of 143 participants, for a prevalence of 28%. Methicillin-resistant S. aureus (MRSA) was carried by 12 participants (8.4%) and methicillin-sensitive S. aureus (MSSA) by 28 (19.6%). The longitudinal study identified 28 (19.6%) persistent carriers, 25 (17.5%) intermittent carriers, and 90 (62.9%) non-carriers. MRSA strains were isolated from 6 persistent carriers and 6 intermittent carriers. Table 1 lists the characteristics of students used in the microbiome study. Underlying data: Table S137 lists the main results obtained from the complete study population.\n\nFigure 1 shows clustering analysis of bacterial genus (A) and fungal species (B) compositions of nasal specimens from the cohort of 15 healthy medical students with known S. aureus carriage patterns. Bacterial microbiome analysis, sequencing, quality filtering and mapping resulted in 1,424,972 mapped V4-region sequences, ranging between 29,971-217,540 copies per sample (average 120,062; SD = 53,027), corresponding to 600 OTUs. We identified 57 of the 600 OTUs (9.5%) at the genus level, while the remaining OTUs mapped to unclassified genera (70.8%) or upper taxonomic groups (96.8%). Three phyla were identified, Proteobacteria being the most abundant (78.3% of 16S rRNA sequences) and the most diverse (42.2% of all identified OTUs), followed by Firmicutes and Actinobacteria. In general, the nasal bacterial microbiome was dominated by the Class Gammaproteobacteria (order Pseudomonadales and genera Citrobacter and Acinetobacter), which is consistent with a 2016-review by Lee et al.38. Table 2 shows the predominant bacterial genera isolated from the nostrils of healthy medical students.\n\nCluster analysis of bacterial genus (A) and fungal species (B) compositions in the nares of healthy medical students with a known S. aureus carriage pattern. Heatmap was constructed using normalized log10 abundance of each OTU in each sample type. Data are presented only for the 20 most abundant taxa. Colored bar above heatmap indicates S. aureus carriage status. P: persistent (red), I: intermittent (blue), NP: non-carrier (green).\n\nAlpha-diversity analysis found that bacterial diversity was greater in the persistent group followed by the intermittent and then by the non-carriers, as shown in Figure 2A. However, the difference in diversity among the three carrier groups was non-significant. Beta-diversity analysis of bacterial communities showed non-significant separation of groups (Figure 2B). Analysis of differential abundance identified that order Pseudomonadales was more abundant in non-carriers followed by intermittent and persistent categories (Figure 3), a finding that may have implications regarding microbial antagonism. However, this trend did not reach statistical significance (p>0.05, Kruskal–Wallis test).\n\n(A) Alpha-diversity, measured by Chao1 and Shannon diversity Index, is plotted for individuals with different S. aureus carrier status: non-carriers (NP, dark green), intermittent carriers (I, purple) and persistent carriers (P, light green). The Chao1 index (left panel) and Shannon index (right panel) were computed for all 15 subjects. The line inside the box represents the median, while the whiskers represent the lowest and highest values within the 1.5 interquartile range (IQR). Statistical testing showed no significant differences among the groups: Chao1 p = 0.0675; Shannon p =0.108. (B) Comparison of beta-diversity of bacterial composition between S. aureus carriage groups with NMDS ordinance calculated from Bray–Curtis distance estimation. I: Intermittent, NP: non-carrier, P: persistent.\n\n(A) Differential abundance at the genus level. (B) Differential abundance for the order Pseudomonadales. Taxa with minimum median abundance of 1% were used for the comparison. There was non-significant difference in abundance among the three carrier groups (p>0.05, Kruskal–Wallis test). NP: non-carrier, I: Intermittent, P: persistent.\n\nFor fungal microbiome analysis, sequencing, quality filtering and mapping resulted in 4,274,743 mapped ITS-region sequences, ranging between 9,543-743,278 copies per sample (average 284,983; SD = 237,339), corresponding to 8,346 fungal OTUs (average 556; SD = 333) (Table 3). We were able to classify 4,453 of the 8,346 OTUs (53.3%) down to species level. Out of the seven recognized major phylum of fungi, three phyla were identified in the nostrils, being Ascomycota the most abundant (90.8% of fungal sequences) and the most diverse (88.7% of all identified OTUs), followed by Basidiomycota and Neocallimastigomycota (containing anaerobic fungi). In general, the nasal mycobiome was dominated by species of the phylum Ascomycota, which is consistent with a 2013-publication by Findley, et al.39. Table 4 shows the predominant fungi species identified from the nostrils of healthy medical students. The most frequent fungal species detected was Penicillium oxalicum, representing 19.0% of the total 4,274,743 sequence reads in the chimera‐filtered OTU table. Malassezia restricta was the second most frequent fungus (12.2% of all sequences). 4.4% of all sequences were unassigned sequences, thought to represent non-fungal contamination.\n\nBased on the identified OTUs and number of sequences in this study, we report a substantial diversity and amount of fungal biomass in the anterior nares of this group of medical students with Colombian ethnicity. Consistent with the 16S analysis, alpha-diversity of fungal communities showed that fungal diversity was greater in the persistent group followed by the intermittent and then by the non-carrier groups. However, this trend did not reach statistical significance (Figure 4A). Beta-diversity analysis of fungal communities showed non-significant separation of groups (Figure 4B).\n\n(A) Alpha-diversity, measured by Chao1 and Shannon diversity Index is plotted for individuals with different S. aureus carrier status: non-carriers (NP, dark green), intermittent carriers (I, purple) and persistent carriers (P, light green). The Chao1 index (left panel) and Shannon index (right panel) were computed for 15 subjects. The line inside the box represents the median, while the whiskers represent the lowest and highest values within the 1.5 interquartile range (IQR). Statistical testing showed no significant differences among the groups: Chao1 p = 0.13; Shannon p = 0.566. (B) Comparison of beta-diversity of fungal composition between S. aureus carriage groups with NMDS ordinance calculated from Bray–Curtis distance estimation. I: Intermittent, NP: non-carrier, P: persistent.\n\nAnalysis of differential abundance showed increased abundance for several fungi species in the persistent group compared to the intermittent and non-carriers (Figure 5A). However, the species Candida orthopsilosis was the only one with significant difference in abundance between the persistent and non-carrier groups (p<0.05, Kruskal-Wallis test) (Figure 5B).\n\n(A) Differential abundance at the species level. (B) Differential abundance for the species Candida orthopsilosis showed a significant difference in abundance between the persistent and non-carrier groups (p<0.05, Kruskal-Wallis test). Taxa with minimum median abundance of 1% were used for the comparison. NP: non-carrier, I: Intermittent, P: persistent.\n\n\nDiscussion\n\nPrevalence of S. aureus nasal carriage was 28%, which is consistent with other studies7. We also found that 37.1% of second-year medical students carried S. aureus in their nares, persistently or intermittently. The distinction in carriage category is important, as persistent carriers are at higher risk of developing active autoinfection than intermittent and non-carriers40–43. MRSA was carried by 8.4% of participants, which represents an important increase from the 1.6% MRSA carriage that we previously reported for our institution in 20126.\n\nWe sought to analyze both bacterial and fungal composition in individuals with a known long-term nasal carriage pattern for S. aureus. We found that nasal bacterial microbiome had low diversity at the phylum level, with three dominating phyla: Proteobacteria, Firmicutes, and Actinobacteria44,45. The top five most abundant genera were Citrobacter, Acinetobacter, Corynebacterium, Paenibacillus and Bacillus, all of which contain pathogenic species, evidencing the potential of the anterior nares as reservoir for pathogens46–48. An interesting finding was that abundance of the genus Staphylococcus in the nares was generally low, even in nasal carriers of S. aureus. The estimated bacterial richness (# of species) found in our study is consistent with a former study that reported an estimate of 2,264 species in the anterior nares based on V3-V5 16S rRNA sequencing49. Recent studies suggest that composition of the nasal microbiota greatly influences S. aureus nasal colonization50,51. However, the mechanisms used by the nasal microbiota to antagonize S. aureus colonization are not completely understood46. Our results are in concordance with those from the Human Microbiome Project in the sense that microbiome composition varies by anatomical site and that interpersonal variation is significant52,53.\n\nRecently, researchers started to focus on the fungal component of the microbiome, revealing the remarkable diversity of the human mycobiome. Fungi were detected at varying abundance in our three carriage groups. The most abundant fungus identified was Penicillium oxalicum, a common environmental fungi that has been recently identified as a cause of invasive mycosis in immunocompromised patients54. Although not statistically significant, we identified a trend towards higher richness and evenness of both bacteria and fungi in the persistent group compared to the intermittent and non-carrier groups. Other studies have suggested that a more diverse microbiota may be associated with resistance to colonization by pathogens; however, we did not observe this phenomenon in our study. Here, a more diverse bacterial and fungal microbiome in the anterior nares seems to favor S. aureus carriage. A similar finding was reported for the pathogen Streptococcus pneumoniae, where a more diverse nasopharyngeal microbiome appeared to facilitate pneumococcal carriage in this human niche55.\n\nWe could not evaluate completely the involvement of specific OTUs in S. aureus carriage due to the small sample size. However, we could identify that the order Pseudomonadales was enriched in non-carriers and that the fungi species Candida orthopsilosis was significantly enriched in the persistent group. These results may suggest that some species in the Pseudomonadales antagonize long-term colonization by S. aureus or that S. aureus colonization may impact the composition of the underlying bacterial communities in the nares, displacing other microbial communities, as has been proposed by others16. Our results also suggest that Candida species may favor the long-term S. aureus colonization of human nares; however, additional studies are required to test these suggestions. Further studies with larger sample size and defined ethnic background are required to identify the interactions between specific members of the resident microbiota that favor or antagonize the colonization process of the bacterium S. aureus in the anterior nares of specific ethnic groups of the human population. This study is the first to analyze simultaneously the bacterial and fungal communities in the nostrils of healthy medical students with a Hispanic/Latino background, and their association with S. aureus nasal colonization.\n\n\nData availability\n\nNasal Microbiome. Raw sequencing data of the nasal microbiome of a set of medical school students, Accession number, PRJNA600228: https://www.ncbi.nlm.nih.gov/bioproject/600228.\n\nOpen Science Framework: Nasal Microbiome of Medical Students UdeC. https://doi.org/10.17605/OSF.IO/UDNWA37.\n\nThis project contains the following underlying data:\n\n- Table S1: Data for complete study population\n\n- JG_16S_OTU_L6: Bacterial species found for microbiome study of 15 participants.\n\n- JG_ITS_OTU_L7: Fungal species found for microbiome study of 15 participants.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
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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\nKluytmans J, van Belkum A, Verbrugh H: Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev. 1997; 10(3): 505–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUsyk M, Zolnik CP, Patel H, et al.: Novel ITS1 Fungal Primers for Characterization of the Mycobiome. mSphere. 2017; 2(6): pii: e00488-17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaporaso JG, Lauber CL, Walters WA, et al.: Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A. 2011; 108 Suppl 1: 4516–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Qian PY: Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One. 2009; 4(10): e7401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmieder R, Edwards R: Quality control and preprocessing of metagenomic datasets. Bioinformatics. 2011; 27(6): 863–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMasella AP, Bartram AK, Truszkowski JM, et al.: PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics. 2012; 13: 31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaporaso JG, Kuczynski J, Stombaugh J, et al.: QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010; 7(5): 335–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010; 26(19): 2460–1. PubMed Abstract | Publisher Full Text\n\nDeSantis TZ, Hugenholtz P, Larsen N, et al.: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006; 72(7): 5069–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang J, Iliev ID, Brown J, et al.: Mycobiome: Approaches to analysis of intestinal fungi. J Immunol Methods. 2015; 421: 112–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRognes T, Flouri T, Nichols B, et al.: VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016; 4: e2584. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbarenkov K, Henrik Nilsson R, Larsson KH, et al.: The UNITE database for molecular identification of fungi--recent updates and future perspectives. New Phytol. 2010; 186(2): 281–5. PubMed Abstract | Publisher Full Text\n\nTeam R: R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. 2018. Reference Source\n\nMcMurdie PJ, Holmes S: phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013; 8(4): e61217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOksanen J, Kindt R, Legendre P, et al.: The vegan package. Community ecology package, R.F.f.S. Computing, Editor. Vienna, Austria. 2007; 10: 631–637. Reference Source\n\nBatdorf C: Coin-package. US. 1903.\n\nWickham H: Reshape2: flexibly reshape data: a reboot of the reshape package. R package version 1. R.F.f.S. Computing, Editor. Vienna, Austria. 2013.\n\nWickham H, Chang W: An implementation of the Grammar of Graphics. R package version. R.F.f.S. Computing, Editor. Vienna, Austria. 2013.\n\nReyes N: Nasal Microbiome of Medical Students Udec. OSF. 2020. http://www.doi.org/10.17605/OSF.IO/UDNWA\n\nLee JT, Frank DN, Ramakrishnan V: Microbiome of the paranasal sinuses: Update and literature review. Am J Rhinol Allergy. 2016; 30(1): 3–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFindley K, Oh J, Yang J, et al.: Topographic diversity of fungal and bacterial communities in human skin. Nature. 2013; 498(7454): 367–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWertheim HF, Melles DC, Vos MC, et al.: The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005; 5(12): 751–62. PubMed Abstract | Publisher Full Text\n\nWertheim HF, Vos MC, Ott A, et al.: Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-carriers. Lancet. 2004; 364(9435): 703–5. PubMed Abstract | Publisher Full Text\n\nNouwen JL, Fieren MW, Snijders S, et al.: Persistent (not intermittent) nasal carriage of Staphylococcus aureus is the determinant of CPD-related infections. Kidney Int. 2005; 67(3): 1084–92. PubMed Abstract | Publisher Full Text\n\nvon Eiff C, Becker K, Machka K, et al.: Nasal carriage as a source of Staphylococcus aureus bacteremia. Study Group. N Engl J Med. 2001; 344(1): 11–6. PubMed Abstract | Publisher Full Text\n\nHuman Microbiome Project Consortium: Structure, function and diversity of the healthy human microbiome. Nature. 2012; 486(7402): 207–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCostello EK, Lauber CL, Hamady M, et al.: Bacterial community variation in human body habitats across space and time. Science. 2009; 326(5960): 1694–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrismer B, Weidenmaier C, Zipperer A, et al.: The commensal lifestyle of Staphylococcus aureus and its interactions with the nasal microbiota. Nat Rev Microbiol. 2017; 15(11): 675–687. PubMed Abstract | Publisher Full Text\n\nSaez-Nieto JA, Medina-Pascual MJ, Carrasco G, et al.: Paenibacillus spp. isolated from human and environmental samples in Spain: detection of 11 new species. New Microbes New Infect. 2017; 19: 19–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan H, Cui B, Huang Y, et al.: Nasal carriage of common bacterial pathogens among healthy kindergarten children in Chaoshan region, southern China: a cross-sectional study. BMC Pediatr. 2016; 16(1): 161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuse SM, Ye Y, Zhou Y, et al.: A core human microbiome as viewed through 16S rRNA sequence clusters. PLoS One. 2012; 7(6): e34242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndersen PS, Pedersen JK, Fode P, et al.: Influence of host genetics and environment on nasal carriage of Staphylococcus aureus in danish middle-aged and elderly twins. J Infect Dis. 2012; 206(8): 1178–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu CM, Price LB, Hungate BA, et al.: Staphylococcus aureus and the ecology of the nasal microbiome. Sci Adv. 2015; 1(5): e1400216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEckburg PB, Bik EM, Bernstein CN, et al.: Diversity of the human intestinal microbial flora. Science. 2005; 308(5728): 1635–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBogaert D, Keijser B, Huse S, et al.: Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis. PLoS One. 2011; 6(2): e17035. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChowdhary A, Kathuria S, Agarwal K, et al.: Voriconazole-Resistant Penicillium oxalicum: An Emerging Pathogen in Immunocompromised Hosts. Open Forum Infect Dis. 2014; 1(2): ofu029. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCremers AJ, Zomer AL, Gritzfeld JF, et al.: The adult nasopharyngeal microbiome as a determinant of pneumococcal acquisition. Microbiome. 2014; 2: 44. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "59469",
"date": "18 Mar 2020",
"name": "Marcelo Brocchi",
"expertise": [
"Reviewer Expertise Microbiology and Genetics of Microorganisms."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study describing the Staphylococcus aureus carrying state among medical students in Colombia. The prevalence of MRSA strains was investigated and the association of the S. aureus carrying state with microbiome composition investigated in a minor number of individuals. It was observed that bacterial and fungal diversity was higher in individuals colonized by S. aureus than in non-carriers although the differences were non-significant. These results contributed with epidemiological information on S. aureus carrier state in Colombian medical students. Maybe, microbiome analysis in a greater number of individuals could reveal a more robust association with S. aureus carrier state. However, the data presented suggest some interesting associations that deserve publication. My only suggestion to the authors is the inclusion of a paragraph in the discussion section about microbiome composition and ethnic groups. Are there differences among Hispanic and other ethnic groups regarding microbiome composition and diversity?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "5414",
"date": "21 Apr 2020",
"name": "NIRADIZ REYES",
"role": "Author Response",
"response": "The authors are thankful for the Reviewer´s recommendations. We have followed them and modified the article accordingly."
}
]
},
{
"id": "61250",
"date": "08 Apr 2020",
"name": "Anne-Sofie Furberg",
"expertise": [
"Reviewer Expertise Infectious disease epidemiology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aim to assess the prevalence of S. aureus nasal carriage and S. aureus nasal carriage phenotypes among medical students in Colombia, and to examine whether S. aureus nasal carriage is associated with composition of the nasal microbiome. S. aureus nasal carriage among medical students and health care workers may represent a source for transmission of the microbe and infection in susceptible patient groups. S. aureus carriage is partly determined by interactions with other members of the microflora. According to the authors, this is the first study analyzing simultaneously the bacterial and fungal communities in the nares of healthy medical students with a Hispanic/Latino background. The study was done among second-year medical students at the same university during a six months period in 2018. The authors collected five repeated nasal swabs for S. aureus culturing from 143 students, and an additional nasal swab for microbiome analysis from a random sample of 15 students having different S. aureus carriage phenotypes. Using only data from the first set of nasal swabs, they report a prevalence of S. aureus carriage of 28%. Using data from all five time points, they report that 20% are persistent carriers. They show that nasal samples from persistent carriers had higher bacterial and fungal diversity than intermittent carriers and non-carriers, however not statistically significant. S. aureus carrier groups could not be separated by bacterial or fungal diversity.\nThe manuscript is generally well written and the results are easy to read from tables and figures. The classification of S. aureus carriage phenotypes by repeated sampling in a relatively large sample is a major strength. The analysis of S. aureus carriage in relation to the microbiome is timely and relevant. However, the present study has some limitations that should be addressed.\nMicrobiome data was collected at the end of the study among 15 students within the three S. aureus carriage phenotype groups. It would be interesting to know more about the stability of the nasal microbiome over time. Did the authors consider taking nasal swabs for microbiome analysis at another time point, e.g. at “baseline” when the first set of swabs for S. aureus culturing were taken? Please, consider rephrasing the sentences in Discussion page 9 “Here, a more diverse…microbiome ..seems to favor S. aureus carriage.” and “Our results also suggest that Candida species may favor the long-term S. aureus colonization of human nares.”, as the design of the present study may not be in line with these interpretations. Please, explain why the microbiome analysis was limited to five students in each group. Did the authors perform any power analysis?\nS. aureus nasal colonization was assessed at five different time points. Could these data have been used to estimate S. aureus prevalence?\nAbstract: The authors may consider a more consistent use of terminology; e.g. patterns of S. aureus nasal carriage or patterns of S. aureus colonization. First part of conclusion can be moved to results.\nIntroduction: The first sentence may be modified as the anterior nares are important due to several functions.\nMethods: Five nasal swab samples for S. aureus culturing were collected with three weeks intervals. Please, describe how information about factors defined in exclusion criteria was collected and updated during the minimum 12 weeks period; e.g. recent infections, antibiotic use. Please, describe the procedure for specimen collection in more detail; i.e. how did the authors standardize the collection method, who did the sampling and where? The nasal swab for microbiome analysis was kept at room temperature and transported to New York, USA, for analysis. Please, include information about transport time and stability of the material from collection to laboratory analysis.\nResults: It would be interesting to know more about the characteristics of the study population for comparison with other studies; e.g. sex, age- and BMI distribution. Results and figures: Please, include “statistically” when referring to the results of statistical tests; i.e. statistically non-significant.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "5415",
"date": "21 Apr 2020",
"name": "NIRADIZ REYES",
"role": "Author Response",
"response": "The authors are thankful for the Reviewer´s recommendations. We greatly value your comments and consider that they have improved the quality of our article. We have followed them and modified the article accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/9-78
|
https://f1000research.com/articles/8-90/v1
|
23 Jan 19
|
{
"type": "Research Article",
"title": "Analysis of CDKN1C in fetal growth restriction and pregnancy loss",
"authors": [
"Jenifer P. Suntharalingham",
"Miho Ishida",
"Federica Buonocore",
"Ignacio del Valle",
"Nita Solanky",
"Charalambos Demetriou",
"Lesley Regan",
"Gudrun E. Moore",
"John C. Achermann",
"Jenifer P. Suntharalingham",
"Miho Ishida",
"Federica Buonocore",
"Ignacio del Valle",
"Nita Solanky",
"Charalambos Demetriou",
"Lesley Regan",
"Gudrun E. Moore"
],
"abstract": "Background: Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a key negative regulator of cell growth encoded by a paternally imprinted/maternally expressed gene in humans. Loss-of-function variants in CDKN1C are associated with an overgrowth condition (Beckwith-Wiedemann Syndrome) whereas “gain-of-function” variants in CDKN1C that increase protein stability cause growth restriction as part of IMAGe syndrome (Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies). As two families have been reported with CDKN1C mutations who have fetal growth restriction (FGR)/Silver-Russell syndrome (SRS) without adrenal insufficiency, we investigated whether pathogenic variants in CDKN1C could be associated with isolated growth restriction or recurrent loss of pregnancy. Methods: Analysis of published literature was undertaken to review the localisation of variants in CDKN1C associated with IMAGe syndrome or fetal growth restriction. CDKN1C expression in different tissues was analysed in available RNA-Seq data (Human Protein Atlas). Targeted sequencing was used to investigate the critical region of CDKN1C for potential pathogenic variants in SRS (n=58), FGR (n=26), DNA from spontaneous loss of pregnancy (n= 21) and women with recurrent miscarriages (n=71) (total n=176). Results: All published single nucleotide variants associated with IMAGe syndrome are located in a highly-conserved “hot-spot” within the PCNA-binding domain of CDKN1C between codons 272-279. Variants associated with familial growth restriction but normal adrenal function currently affect codons 279 and 281. CDKN1C is highly expressed in the placenta compared to adult tissues, which may contribute to the FGR phenotype and supports a role in pregnancy maintenance. In the patient cohorts studied no pathogenic variants were identified in the PCNA-binding domain of CDKN1C. Conclusion: CDKN1C is a key negative regulator of growth. Variants in a very localised “hot-spot” cause growth restriction, with or without adrenal insufficiency. However, pathogenic variants in this region are not a common cause of isolated fetal growth restriction phenotypes or loss-of-pregnancy/recurrent miscarriages.",
"keywords": [
"CDKN1C",
"intra-uterine growth restriction",
"fetal growth restriction",
"Silver-Russell syndrome",
"IMAGe syndrome",
"adrenal",
"placenta",
"recurrent miscarriage"
],
"content": "Introduction\n\nCyclin-dependent kinase inhibitor 1C (CDKN1C, also known as P57/kip2) (OMIM 600856) is a key negative regulator of cell proliferation that is encoded by a paternally imprinted (maternally expressed) gene on the short arm of chromosome 11 (11p15.4) in humans (Stampone et al., 2018).\n\nConsistent with its role in growth and development, maternally-inherited loss-of-function variants in CDKN1C are found in approximately 5–10% of individuals with the “overgrowth” condition, Beckwith-Wiedemann Syndrome (BWS) (OMIM 130650) (Eggermann et al., 2014). Clinical features of BWS include macrosomia, hyperinsulinism and adrenal tumors.\n\nIn contrast, gain-of-function variants in CDKN1C have been shown to cause growth restriction as part of IMAGe syndrome (OMIM 614732) (Arboleda et al., 2012). IMAGe syndrome is characterised by fetal/Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies (in males, usually relatively mild hypospadias and undescended testes) as well as additional features such as hearing loss and hypercalciuria (Bennett et al., 1993; Vilain et al., 1999).\n\nTo date, children with IMAGe syndrome have all been found to harbour pathogenic single nucleotide variants (SNVs) in a very specific region of the PCNA-binding domain of CDKN1C (Arboleda et al., 2012; Cabrera-Salcedo et al., 2017). These changes potentially lead to increased activity through increasing protein stability, thereby preventing cell cycle progression into S phase (Borges et al., 2015; Hamajima et al., 2013).\n\nMore recently, SNVs in the PCNA-binding domain of CDKN1C have been reported in families with maternally-inherited fetal growth restriction (FGR) without adrenal insufficiency and in familial Silver-Russell syndrome (SRS) (OMIM 180860) (Brioude et al., 2013; Kerns et al., 2014). SRS is characterised by variable clinical features including fetal and post-natal growth restriction, relative macrocephaly, feeding difficulties and characteristic facies. SRS is also described as phenotypically and genotypically opposite to BWS and approximately half of the molecular anomalies are attributed to Chr11p15.5 imprinting clusters, including several individuals with maternal duplication of the locus containing CDKN1C (Bonaldi et al., 2011; Boonen et al., 2016; Schönherr et al., 2007). These findings suggest that the growth restriction phenotype associated with CDKN1C may be more variable and adrenal insufficiency is not always present.\n\nThe aim of this study was therefore to review published CDKN1C variants associated with FGR/IUGR phenotypes, to study CDKN1C expression in different tissues, and to analyse the critical region in CDKN1C in a range of growth restriction and adverse pregnancy phenotypes, with a hypothesis that severe restriction of feto-placental growth may, in some situations, result in pregnancy loss or recurrent miscarriage.\n\n\nMethods\n\nA PubMed search was undertaken (March 2018) using the search terms “CDKN1C”, “human” and “growth”, or “IMAGe syndrome”. Reports focusing on growth restriction phenotypes associated with single nucleotide variants were considered. Population variation in CDKN1C was assessed using the gnomAD browser (http://gnomad.broadinstitute.org; accessed April 2018) (Lek et al., 2016). Protein conservancy analysis was performed using ClustalW in Jalview (Waterhouse et al., 2009).\n\nRNA-Seq data for CDKN1C expression was obtained with specific permission from the Human Protein Atlas (Human Protein Atlas available from www.proteinatlas.org) and re-drawn in R (version 3.4.2) (Uhlen et al., 2015).\n\nThe following growth restriction cohorts were included in this study: 1) SRS (n=58) (isolated, non-familial) diagnosed on consensus criteria and where maternal uniparental disomy or H19/IGF2:IG-DMR (also known as ICR1 or IC) hypomethylation had been excluded (Wakeling et al., 2017); 2) FGR (n=26) (isolated, non-familial) defined as birth weight less than the 3rd centile, as part of the Baby Bio Bank cohort (UCL-GOS Institute of Child Health & St Mary’s Imperial College London) (Leon et al., 2016). Additional analysis was undertaken in DNA from 3) products of conception (POC) (n=21) where there had been a spontaneous loss of pregnancy and 4) mothers who had a history of recurrent miscarriages (n=71) (at least three miscarriages) where an underlying cause was not known (Baby Bio Bank). An overview of these cohorts is provided in Table 1.\n\nAbbreviations: FGR, fetal growth restriction; IUGR, intrauterine growth restriction\n\nEthical Committee approval for the Baby Bio bank was obtained from the Trent Derby Ethics Committee (09/H0405/30) and Ethical Committee approval for the Silver Russell trios was from GOSH Research Ethics Committee (REC No. 1278). Written informed consent was obtained from participants or parents. DNA was extracted from blood lymphocytes, placental tissue or products of conception, as appropriate.\n\nDirect Sanger Sequencing was undertaken for SRS patients to analyse the PCNA-binding region (codons 213–316) and hotspot (codons 272–281) using primers reported previously (CCDS7738, ENST00000414822.8) (Arboleda et al., 2012). Additional primers pairs were used to sequence the 3’ end of exon 1 and splice site (CDKN1CF: CAGGAGCCTCTCGCTGAC; CDKN1CR2: GCTGGAGGGCACAACAAC). Polymerase chain reaction (PCR) was carried out with BIOTAQ DNA Polymerase (BIOLINE, London, UK). PCR products were purified by microclean (Microzone, Haywards Heath, UK) and amplified with BigDyeTerminator v1.1, followed by sequencing on a DNA Analyzer 3070 (Applied Biosystems, California, US). The resulting read-outs were reviewed in Sequencher (v5.3: Gene Codes).\n\nTargeted array capture followed by next-generation sequencing was performed for FGR samples, products of conception and mothers with a history of recurrent miscarriage.\n\nA targeted enrichment custom HaloPlex 501kb–2.5Mb HS panel (Agilent Technologies Inc.) was designed using Agilent SureDesign to capture known and candidate genes for fetal growth disruption, including CDKN1C. This study used designs targeting either 147 (design size 1.391 Mbp) or 257 (design size 2.045 Mbp) genes.\n\nSequencing libraries were prepared using 50ng of genomic DNA following the manufacturer’s protocol (HaloPlex HS Target Enrichment System for Illumina Sequencing version C1 from December 2016) and as described in principle previously (Guran et al., 2016). This was followed by 2 x 100bp or 2 x 149bp paired end sequencing to a median read depth of 300x on a NextSeq sequencer (Illumina Inc.). The bcl files were converted to fastq files using manufacturers recommended guidelines and were then analysed in SureCall software (version 4.0.1.46 (Agilent Technologies) using the HaloPlex Default Method or custom settings (minimum number of read pairs per barcode =1). Samples with a minimum read depth less than 4 at any single nucleotide position were excluded.\n\n\nResults\n\nReview of available data revealed six publications describing isolated individuals (7) or families (4) with IMAGe syndrome and adrenal insufficiency, who had pathogenic variants in a key region of the PCNA-binding domain of CDKN1C affecting codons 272, 274, 276, 278 and 279 (Figure 1, Table 2) (Arboleda et al., 2012; Bodian et al., 2014; Brioude et al., 2013; Hamajima et al., 2013; Kato et al., 2014; Kerns et al., 2014). These codons are highly conserved amongst species (Figure 2). Multiple individuals from different ancestral backgrounds were found to have p.Asp274Asn or p.Lys278Glu changes.\n\nAbbreviations: DM, diabetes mellitus; IGT, impaired glucose tolerance; IMAGe, intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita, genital anomalies; IUGR, intrauterine growth restriction.\n\nRed arrowheads represent codons that are mutated in IMAGe syndrome, FGR/IUGR or Silver-Russell syndrome. Yellow asterisks represent complete conservation amongst the species shown.\n\nVariants in CDKN1C associated with familial Silver-Russell syndrome or growth restriction but normal adrenal function were found towards the carboxyl-terminal region of this “hot-spot” domain (p.Arg279Leu, p.Arg281Ile) (Figure 1).\n\nAnalysis of population data from the gnomAD browser showed a complete absence of variants in the key codons listed. Very rare heterozygous SNVs were found that are predicted to cause p.Ala277Val (11:2905355G>A;1 in 107288 alleles) and p.Ala283Val (11:2905337G>A; rs776541692; 1 in 30726 alleles) changes. Of note, these codons are two of the lesser-conserved amino acids within this “hot-spot” region (Figure 2).\n\nRNA-Seq analysis of CDKN1C in a panel of human tissues showed highest expression in the placenta (Figure 3; Human Protein Atlas data, https://www.proteinatlas.org/ENSG00000129757-CDKN1C/tissue), with strong expression also in adipose tissue, ovary, adrenal, endometrium and kidney. Immunohistochemistry in the Human Protein Atlas repository shows strong staining in the nuclei of both decidual and trophoblastic cells (https://www.proteinatlas.org/ENSG00000129757-CDKN1C/tissue/placenta).\n\nData reproduced and modified with permission from the Human Protein Atlas (www.proteinatlas.org) (Uhlen et al., 2015). TPM = Transcripts Per Million.\n\nAnalysis of the PCNA-binding domain of CDKN1C by Sanger sequencing in a cohort of 58 children with isolated (non-familial) Silver-Russell syndrome did not reveal any pathogenic variants. Sequencing data for exon 2 is shown in Supplementary Data 1 (Achermann, 2018).\n\nA next-generation sequencing approach of CDKN1C in children with IUGR/FGR (n=26), products of conception (n=21), and mothers with a history of recurrent miscarriage (n=71) also did not reveal pathogenic variants in this region (Table 1). Details of coverage for each sample/cohort and each nucleotide is shown in Supplementary Data 2–7 (Achermann, 2018).\n\n\nDiscussion\n\nCDKN1C is now well-established as a key regulator of cell cycle and growth through G1 phase cell cycle arrest. Although loss-of-function of CDKN1C is known to cause macrosomia as part of Beckwith-Wiedemann Syndrome, it is only in the past six years that “gain-of-function” variants in CDKN1C have been shown to cause growth restriction and IMAGe syndrome. These findings demonstrate clearly how opposite effects in protein function can have opposite phenotypes (Arboleda et al., 2012; Eggermann et al., 2014). Sometimes these features affect not just growth but also endocrine systems (e.g. adrenal tumours (BWS)/adrenal hypoplasia (IMAGe); congenital hyperinsulinism (BWS)/diabetes mellitus (one family with growth restriction)).\n\nReview of published literature confirms that CDKN1C SNVs associated with IMAGe syndrome or growth restriction are all located within a “hot-spot” of the PCNA-binding domain of the protein. The exact function of this region is not clear, and the crystal structure of this region of CDKN1C has not yet been solved. Studies to date suggest pathogenic variants may increase protein stability or reduce the rate of degradation, thereby enhancing the negative effects of CDKN1C on cell cycle progression (Borges et al., 2015; Hamajima et al., 2013). The very localised nature of these variants clearly demonstrates a key role for this region in CDKN1C function.\n\nCDKN1C is strongly expressed in fetal adrenal development as shown by qPCR and immunohistochemistry (Arboleda et al., 2012), by microarray (Del Valle et al., 2017), and RNA-Seq (unpublished). Furthermore, analysis of RNA-Seq data from the Human Protein Atlas shows marked expression in the placenta (Figure 3). Immunohistochemistry shows strong nuclear staining in both decidual and trophoblastic cells. Therefore, gain-of-function of CDKN1C in the developing placenta could have a significant contribution to the growth restriction phenotype.\n\nFurther evidence for a potential role for CDKN1C in fetal growth restriction phenotypes has emerged with reports of CDKN1C variants in familial growth restriction and familial SRS (Brioude et al., 2013; Kerns et al., 2014). Several features of IMAGe syndrome and SRS overlap, such as bi-frontal bossing and relative micrognathia. To date these children have not shown evidence of adrenal insufficiency. Whilst a lack of adrenal features could be due to underlying mosaicism or a somatic “rescue” event, as recently reported for variants in SAMD9 in the related condition MIRAGE syndrome (Buonocore et al., 2017; Narumi et al., 2016), the fact that several individuals in a family with the same CDKN1C SNV were affected provides strong evidence that the primary genomic event is influencing the phenotype rather than a rescue mechanism. Of note, review of the two SNVs associated with FGR and normal adrenal function reveals that they affect amino acids at the C-terminal region of the hotspot. In one family a charged arginine at codon 279 is replaced by a non-polar leucine, whereas a proline at this position is found in classic IMAGe syndrome. In another family, the arginine at position 281 is replaced by a non-polar isoleucine. In some situations, variants in amino-acids flanking critical motifs can be associated with milder phenotypes (Kyriakakis et al., 2017).\n\nDespite these findings, we did not identify any CDKN1C variants in children with isolated (non-familial) SRS or IUGR/FGR. These results are similar to data from Brioude et al. who only reported a CDKN1C variant in familial SRS and did not find variants in 68 children with an isolated condition (Brioude et al., 2013). Whilst this does not exclude CDKN1C as a potential cause of isolated or sporadic SRS/FGR, it does suggest that it is not a common cause.\n\nIdentification of such strong expression of CDKN1C in the placenta lead us to consider whether gain-of-function/increased stability of CDKN1C could be associated with loss of pregnancy or recurrent miscarriage. This hypothesis was supported further by recent studies linking placental genes to pregnancy loss (Perez-Garcia et al., 2018); a potential role for placental CDKN1C expression in fetal growth and regulation by oestrogen (Chen et al., 2018; Gou et al., 2017; López-Abad et al., 2016; Unek et al., 2014); and the fact that paternal imprinting of the gene means that a pool of deleterious variants could be present in the population and cause pregnancy loss (together with live births) in women who carry this variant and inherit it from their father.\n\nAnalysis was therefore undertaken in a cohort of mothers who had recurrent miscarriages (often together with live birth(s)) and also from products of conception. However, no variants were found in the PCNA binding domain of CDKN1C in these cohorts.\n\nThis work has several limitations. Although 176 total individuals were studied, each sub group is still relatively small and rare CDKN1C variants might be discovered if the sample size is increased. The causes of recurrent miscarriage and fetal growth restriction are clearly diverse and can be influenced by many factors, and regulatory regions or enhancers (Perez-Garcia et al., 2018; Rai & Regan, 2006; Stalman et al., 2018; Wakeling et al., 2017). Nevertheless, this study does highlight the role of the key region of CDKN1C in human fetal growth restriction phenotypes, and starts to address the potential role of single gene growth restriction phenotypes in more common obstetric and fetal conditions.\n\n\nData availability\n\nData has been uploaded to OSF: http://doi.org/10.17605/OSF.IO/Y7KZV (Achermann, 2018). Representative sequencing data for chromatograms is shown in Supplementary Data 1. Data of coverage for next generation sequencing is shown in Supplementary Data 2–7.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nAccess for samples from the Baby Bio Bank is available and can be requested from the steering committee by emailing the Baby Bio Bank Manager (nita.solanky@ucl.ac.uk). More information about data access can be found on the website under the ‘Protocol for the management of the Baby Bio Bank’, section 15: http://www.ucl.ac.uk/tapb/sample-and-data-collections-at-ucl/biobanks-ucl/baby-biobank/baby-biobank-documents/BBB_SOP_version7. An application form for the use of Baby Bio Bank resources must be completed prior to application (see appendix 2 in the protocol).",
"appendix": "Grant information\n\nJ.C.A. is a Wellcome Trust Senior Research Fellow in Clinical Science (grant 209328/Z/17/Z) with research support from Great Ormond Street Hospital Children's Charity (grant V2518) and the NIHR GOSH BRC (IS-BRC-1215-20012). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. The Baby Bio Bank was funded by Wellbeing of Women. M.I. and C.D. are funded by the UK MRC.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAchermann J: Analysis of CDKN1C in Fetal Growth Restriction and Pregnancy Loss. Open Science Framework. 2018. http://www.doi.org/10.17605/OSF.IO/Y7KZV\n\nArboleda VA, Lee H, Parnaik R, et al.: Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome. Nat Genet. 2012; 44(7): 788–792. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBennett J, Schrier Vergano SA, Deardorff MA: IMAGe Syndrome. GeneReviews®. 1993; (Accessed: 25 April 2018). PubMed Abstract\n\nBodian DL, Solomon BD, Khromykh A, et al.: Diagnosis of an imprinted-gene syndrome by a novel bioinformatics analysis of whole-genome sequences from a family trio. Mol Genet Genomic Med. 2014; 2(6): 530–538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonaldi A, Mazzeu JF, Costa SS, et al.: Microduplication of the ICR2 domain at chromosome 11p15 and familial Silver-Russell syndrome. Am J Med Genet A. Wiley-Blackwell, 2011; 155A(10): 2479–2483. PubMed Abstract | Publisher Full Text\n\nBoonen SE, Freschi A, Christensen R, et al.: Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes. Clin Epigenetics. 2016; 8(1): 69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorges KS, Arboleda VA, Vilain E: Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase. Cell Div. 2015; 10(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrioude F, Oliver-Petit I, Blaise A, et al.: CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome. J Med Genet. 2013; 50(12): 823–830. PubMed Abstract | Publisher Full Text\n\nBuonocore F, Kühnen P, Suntharalingham JP, et al.: Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans. J Clin Invest. 2017; 127(5): 1700–1713. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCabrera-Salcedo C, Kumar P, Hwa V, et al.: IMAGe and Related Undergrowth Syndromes: The Complex Spectrum of Gain-of-Function CDKN1C Mutations. Pediatr Endocrinol Rev. 2017; 14(3): 289–297. PubMed Abstract\n\nChen XJ, Chen F, Lv PP, et al.: Maternal high estradiol exposure alters CDKN1C and IGF2 expression in human placenta. Placenta. 2018; 61: 72–79. PubMed Abstract | Publisher Full Text\n\nDel Valle I, Buonocore F, Duncan AJ, et al.: A genomic atlas of human adrenal and gonad development [version 2; referees: 4 approved]. Wellcome Open Res. 2017; 2: 25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEggermann T, Binder G, Brioude F, et al.: CDKN1C mutations: two sides of the same coin. Trends Mol Med. 2014; 20(11): 614–622. PubMed Abstract | Publisher Full Text\n\nGou C, Liu X, Shi X, et al.: Placental Expressions of CDKN1C and KCNQ1OT1 in Monozygotic Twins with Selective Intrauterine Growth Restriction. Twin Res Hum Genet. 2017; 20(5): 389–394. PubMed Abstract | Publisher Full Text\n\nGuran T, Buonocore F, Saka N, et al.: Rare Causes of Primary Adrenal Insufficiency: Genetic and Clinical Characterization of a Large Nationwide Cohort. J Clin Endocrinol Metab. 2016; 101(1): 284–292. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamajima N, Johmura Y, Suzuki S, et al.: Increased protein stability of CDKN1C causes a gain-of-function phenotype in patients with IMAGe syndrome. PLoS One. Edited by He B, 2013; 8(9): e75137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKato F, Hamajima T, Hasegawa T, et al.: IMAGe syndrome: clinical and genetic implications based on investigations in three Japanese patients. Clin Endocrinol (Oxf). 2014; 80(5): 706–713. PubMed Abstract | Publisher Full Text\n\nKerns SL, Guevara-Aguirre J, Andrew S, et al.: A novel variant in CDKN1C is associated with intrauterine growth restriction, short stature, and early-adulthood-onset diabetes. J Clin Endocrinol Metab. 2014; 99(10): E2117–E2122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKyriakakis N, Shonibare T, Kyaw-Tun J, et al.: Late-onset X-linked adrenal hypoplasia (DAX-1, NR0B1): two new adult-onset cases from a single center. Pituitary. 2017; 20(5): 585–593. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLek M, Karczewski KJ, Minikel EV, et al.: Analysis of protein-coding genetic variation in 60,706 humans. Nature. Nature Publishing Group, 2016; 536(7616): 285–291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeon LJ, Solanky N, Stalman SE, et al.: A new biological and clinical resource for research into pregnancy complications: The Baby Bio Bank. Placenta. 2016; 46: 31–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLópez-Abad M, Iglesias-Platas I, Monk D: Epigenetic Characterization of CDKN1C in Placenta Samples from Non-syndromic Intrauterine Growth Restriction. Front Genet. 2016; 7: 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNarumi S, Amano N, Ishii T, et al.: SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7. Nat Genet. 2016; 48(7): 792–797. PubMed Abstract | Publisher Full Text\n\nPerez-Garcia V, Fineberg E, Wilson R, et al.: Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature. 2018; 555(7697): 463–468. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRai R, Regan L: Recurrent miscarriage. Lancet. 2006; 368(9535): 601–11. PubMed Abstract | Publisher Full Text\n\nSchönherr N, Meyer E, Roos A, et al.: The centromeric 11p15 imprinting centre is also involved in Silver-Russell syndrome. J Med Genet. BMJ Publishing Group Ltd, 2007; 44(1): 59–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStalman SE, Solanky N, Ishida M, et al.: Genetic Analyses in Small-for-Gestational-Age Newborns. J Clin Endocrinol Metab. 2018; 103(3): 917–925. PubMed Abstract | Publisher Full Text\n\nStampone E, Caldarelli I, Zullo A, et al.: Genetic and Epigenetic Control of CDKN1C Expression: Importance in Cell Commitment and Differentiation, Tissue Homeostasis and Human Diseases. Int J Mol Sci. 2018; 19(4): pii: E1055. PubMed Abstract | Publisher Full Text\n\nUhlen M, Fagerberg L, Hallström BM, et al.: Proteomics. Tissue-based map of the human proteome. Science. 2015; 347(6220): 1260419. PubMed Abstract | Publisher Full Text\n\nUnek G, Ozmen A, Ozekinci M, et al.: Immunolocalization of cell cycle proteins (p57, p27, cyclin D3, PCNA and Ki67) in intrauterine growth retardation (IUGR) and normal human term placentas. Acta Histochemica. 2014; 116(3): 493–502. PubMed Abstract | Publisher Full Text\n\nVilain E, Le Merrer M, Lecointre C, et al.: IMAGe, a new clinical association of intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies. J Clin Endocrinol Metab. 1999; 84(12): 4335–4340. 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}
|
[
{
"id": "50625",
"date": "18 Jul 2019",
"name": "Flavia Cerrato",
"expertise": [
"Reviewer Expertise Genomic imprinting",
"Imprinting disorders"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper Suntharalingham and coworkers investigate the involvement of CDKN1C, a negative regulator of cell proliferation and growth, in fetal growth restriction (FGR)/intrauterine growth restriction (IUGR) human conditions, pregnancy loss or recurrent miscarriage. While many loss-of-function variants have been reported associated with the overgrowth condition Beckwith-Wiedemann syndrome and affecting all the domains of the protein, only few variants predicted to be “gain of function”, have been found associated with syndromic growth restriction conditions (IMAGe and SRS). The authors review the latter cases and find that all reported variants, associated with growth restriction, are localized in the same region inside the PCNA-binding domain, and affect evolutionary conserved amino acids.\n\nBy studying RNA-seq data available on the Human Protein Atlas website, the authors consider the high expression level of CDKN1C in placenta to hypothesize its involvement in pregnancy loss and miscarriage as a consequence of placenta restriction. Following a DNA targeted sequencing analysis restricted to the “hot spot” PCNA-binding domain in isolated cases of SRS (n=58), IUGR/FGR (n=26), loss of pregnancy (n=21) and women with recurrent miscarriage (n=71), they do not find any pathogenic variant, concluding that pathogenic variants of this analysed region are not a common cause of these pathological conditions.\n\nThe paper is interesting and easy to read. The potential role of CDKN1C in pregnancy maintenance and its putative involvement in pregnancy loss is interesting, and an aspect that in future could be better investigated by extending the number of the cases to analyze.\n\nIt would be interesting if the authors could report the analysis of the variants extended to the CDKN1C domains other than the region corresponding to the “hot spot” PCNA-binding domain (at least for cases analysed by NGS).\n\nMinor comments:\nThe authors should clarify why they have analyzed CDKN1C variants only in the isolated SRS cases, excluding the familial cases.\n\nMost of information reported in Table 2 are already present in Figure 1, therefore Table 2 could be removed or reported as Supplementary Table.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5410",
"date": "21 Apr 2020",
"name": "John Achermann",
"role": "Author Response",
"response": "The paper is interesting and easy to read. The potential role of CDKN1C in pregnancy maintenance and its putative involvement in pregnancy loss is interesting, and an aspect that in future could be better investigated by extending the number of the cases to analyze. Response: In the revised submission, we have used the Agilent HaloPlex panel, as well as a Nonacus 3T cell target enrichment approach to add data from an additional 27 subjects (POC (n= 1); women with history of recurrent miscarriage (n=7); Silver Russell Syndrome (n=8) and fetal growth restriction (n=11) ). We have modified Table 1, Supplementary data and Figures, and relevant places in the text, accordingly, and included a Supplementary Data 8 for Nonacus coverage. A reference for the Nonacus methods in included (Buonocore et al., 2019). It would be interesting if the authors could report the analysis of the variants extended to the CDKN1C domains other than the region corresponding to the “hot spot” PCNA-binding domain (at least for cases analysed by NGS). Response: Although our focus was on the PCNA-binding domain, we agree it is valid to look at regions outside of the hotspot and thank the reviewer (and reviewer 2) for the suggestion.Analysis of CDKN1C using the gnomAD browser (accessed March 2020) shows an expected number of synonymous changes (o/e =1.06 (0.86-1.31)) but fewer missense (o/e= 0.51 (0.42-0.63)) and pLoF changes (o/e=0 (0-0.53)) than expected. (https://gnomad.broadinstitute.org/gene/ENSG00000129757?dataset=gnomad_r2_1)Based on the fact that random variants within a gene are more likely to cause disruption rather than gain-of-function, the low number of observed missense changes is more likely to represent an intolerance to disruption and loss of function associated with overgrowth and Beckwith-Wiedemann Syndrome, rather than rare gain of function changes associated with FGR/IMAGe syndrome. We have undertaken an extended analysis of data for the gene. We did not detect any category 1 variants (nonsense, frameshift, canonical splice site variants) at all, which is not surprising, as these would be loss of function and predicted to be associated with overgrowth.We only identified two category 2 (non-synonymous missense) variants in the cohort, both in women who have had recurrent miscarriages. One of these is a 9:g.2906703A>G variant (rs201715947) predicted to result in a p.Leu6Pro change that is present in gnomAD with allelic frequency of 0.0001102 (8/72604). The other variant is a 9:g.2906589C>T predicted to result in a p. Arg44His. This variant is not present in gnomAD but a p.Arg44Leu is present in one male. Although the SIFT score for this variant suggests it is deleterious, we find that prediction models are not always reliable for gain-of-function effects. On balance, we do not feel that either of these variants are likely to be clinically pathogenic although we cannot say this definitively. We have commented on this in the revised results. More certain is our finding that there is not a large pool of additional CDKN1C variants in our cohort outside of the PCNA binding domain.Minor comments:• The authors should clarify why they have analyzed CDKN1C variants only in the isolated SRS cases, excluding the familial cases.Response: Our cohort was only made up of sporadic Silver Russell Syndrome subjects; we had no familial cases. We have mentioned this in the methods. • Most of information reported in Table 2 are already present in Figure 1, therefore Table 2 could be removed or reported as Supplementary Table 2.Response: We agree there is some overlap, but we would prefer to keep both in the main body of text as Figure 1 shows the structure of CDKN1C and position of variants, and Table 2 shows nucleotide data, whether mineralocorticoid or glucocorticoid was given, and links to the original references."
}
]
},
{
"id": "51957",
"date": "27 Aug 2019",
"name": "Amanda J. Drake",
"expertise": [
"Reviewer Expertise Epigenetics",
"paediatrics",
"fetal growth",
"obstetrics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report Suntharalingham and colleagues investigate the extent to which variations in a known negative regulator of cell growth (CDKN1C) may be involved in isolated growth restriction or recurrent pregnancy loss.\n\nThey approach this in several ways:\nThrough analysis of published literature\n\nThrough analysis of CDKN1C expression in multiple tissues using available RNA-seq data\n\nTargeted sequencing in a number of patient populations.\nAs a result of the literature search, they conclude that reported variants are located in the PCNA-binding domain of CDKN1C and therefore focus on this for their sequencing analysis. The analysis of available RNA-seq data suggests that the gene is highly expressed in placenta, suggesting it may have a role in pregnancy maintenance. Sequencing analysis revealed no pathogenic variants in the targeted region of CDKN1C suggesting this is an uncommon cause of fetal growth restriction or recurrent pregnancy loss.\nThe paper is well written and easy to read, with appropriate citation of the literature.\nMinor comments:\nPubMed search terms appear quite limited. Are the authors certain they have not missed any papers by not including terms such as IUGR, FGR, SGA?\n\nDo the pregnancies/children in the FGR/IUGR group have any other complications?\n\nThe term 'usually a history of live births' in table 2 is rather vague. Were any of these live births also affected by FGR/IUGR etc. or by pregnancy complications commonly associated with poor fetal growth e.g. pre-eclampsia.\n\nIn table 2 - do the numbers in brackets refer to the number of families with that variant or the number of affected individuals within a family?\nGiven the limited number of publications describing pathogenic mutations in the PCNA-binding domain of CDKN1C in IMAGe syndrome, familial SRS and in a single family with FGR and early adult onset diabetes, are the researchers planning to analyse other regions of the gene/its regulatory regions for other variations which may affect expression? Could they take this on to include analysis of placental gene expression/protein expression/function in assocision with analysis of sequence variation?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5411",
"date": "21 Apr 2020",
"name": "John Achermann",
"role": "Author Response",
"response": "Minor comments:1. PubMed search terms appear quite limited. Are the authors certain they have not missed any papers by not including terms such as IUGR, FGR, SGA?Response: Thank you for this comment. We have used those additional search terms and not found any additional publications. We have modified the methods accordingly. A more recent search of CDKN1C and IMAGe has revealed an interesting additional family of two half-siblings with Silver Russell Syndrome (SRS) (Sabir et al 2019), who have a c.836 G> T variant resulting in the p.Arg279Leu change previously reported by Brioude et al. We have modified Figure 1 and Table 2 to include these important new data, which supports the hypothesis that the p.Arg279Leu change (at the edge of the PCNA-binding domain) is associated with SRS, rather than the more severe IMAGe phenotype. We have added this new reference to the paper and clarified the footnote in Table 2 to show that the numbers in parentheses refer to numbers of affected individuals within each kindred.2. Do the pregnancies/children in the FGR/IUGR group have any other complications?Response: To our knowledge this group do not have additional multisystem features.3. The term 'usually a history of live births' in table 2 is rather vague. Were any of these live births also affected by FGR/IUGR etc. or by pregnancy complications commonly associated with poor fetal growth e.g. pre-eclampsia. Response: This is a good point. Unfortunately, we do not have easy access to those data at present.4. In table 2 - do the numbers in brackets refer to the number of families with that variant or the number of affected individuals within a family?Response: Thank you for pointing this out. he number refers to the number of affected individuals within a family. We have added a footnote to clarify this. Given the limited number of publications describing pathogenic mutations in the PCNA-binding domain of CDKN1C in IMAGe syndrome, familial SRS and in a single family with FGR and early adult onset diabetes, are the researchers planning to analyse other regions of the gene/its regulatory regions for other variations which may affect expression? Could they take this on to include analysis of placental gene expression/protein expression/function in association with analysis of sequence variation?Response: We have analysed the rest of CDKN1C beyond the PCNA-binding domain wherever possible and did not find any variants that are clearly pathogenic (see response to reviewer 1). We did not build promoter or regulatory regions of CDKN1C into our capture design but it is an interesting point and maybe this can be addressed as more whole genome sequencing data become available. As pointed out, correlating genomic variations with placental gene and protein expression in the same individual would be a very powerful approach, if appropriate samples were available."
}
]
}
] | 1
|
https://f1000research.com/articles/8-90
|
https://f1000research.com/articles/9-269/v1
|
17 Apr 20
|
{
"type": "Research Article",
"title": "Gender-based violence: Experiences from two tertiary care settings in Sri Lanka",
"authors": [
"Dasuni Yahanika Pathiraja",
"Ramya Priyanwada Pathiraja",
"Lakshmen Senanayake",
"Rukshani Mayawanthi Edirisinghe",
"Nethanjalie Mapitigama",
"Dasuni Yahanika Pathiraja",
"Lakshmen Senanayake",
"Rukshani Mayawanthi Edirisinghe",
"Nethanjalie Mapitigama"
],
"abstract": "Background: This study aimed to obtain an overview of survivors of gender-based violence GBV who seek care, different types and consequences of (GBV), their modes of referral, factors associated with GBV, characteristics of the perpetrators, health-seeking behavior of the care-seekers and the service provided by GBV Care Centers in two tertiary care settings Methods: A retrospective cross-sectional study was conducted from January 2017 to December 2019 at two GBV care centers in a Women’s Hospital and a General Hospital in Colombo, Sri Lanka. Sociodemographic details of care-seekers, referral methods, types of violence experienced and their consequences, factors associated with GBV, characteristics of the perpetrator, health seeking behavior of those seeking care, and the services provided, were obtained from the hospital records. Results: Records from all care seekers (n=495 women, no men) were obtained, and 488 were suitable for analysis. More women presented with GBV to the Women’s Hospital compared to the General Hospital (395 vs 93, p<0.001), and there were significant differences in modes of referral between the two hospitals. A large majority had suffered emotional and economic violence, although physical or sexual violence were the reasons for referral to the centers. Suicidal tendencies had been reported by 20%. In 94.2% of cases the husband, lover or partner was the perpetrator. Physical violence was more likely in married women, those who did not report a stable relationship, and in those who were employed. Of the 488 women, 37% were pregnant at the time of violence. Most of the women had confided with another female about the violence. Less than 5% came for follow-up. Conclusions: GBV care services should be offered in all hospitals, especially those providing maternity and gynaecological care. Emotional and economic violence are common but often overlooked. There is a need to increase public awareness about GBV.",
"keywords": [
"Gender-based violence",
"tertiary care settings",
"Sri Lanka"
],
"content": "Introduction\n\nGender-based violence (GBV) is one of the most notable human rights violations that occurs in most societies. Both women and men experience GBV, but the majority of survivors are women and girls1. GBV could be perpetrated in multiple ways, involving sexual, physical, emotional and economical and social dimensions2. GBV, including intimate partner violence (IPV) is pervasive globally and leads to significant physical and mental health problems among survivors and their children3. Intimate partner violence can have an influence on the behavior of the next generation4.\n\nIn the Demographic and Health Survey of Sri Lanka in 2016, a prevalence of 17% of GBV was found among ever-married women of the 15–49 age group, with a prevalence of 19.3%, 17% and 16% from the urban, estate, and rural sectors, respectively5. Physical abuse during current pregnancy was reported as 4.7% in a study among 1200 women in Badulla District in 20046. Intimate partner violence in pregnancy is known to have many negative outcomes, including miscarriages, still births and maternal deaths7.\n\nGender and Women’s Health Unit of the Family Health Bureau of the Ministry of Health, Sri Lanka is responsible for directing the health sector response at national level. The Gender and Women’s health Unit has established dedicated GBV care centers under the name of “Mithuru Piyasa” / Natpu Nilayam, meaning the Friendly Haven, within the network of hospitals spread throughout the country to provide services to survivors of GBV. There are 70 centers spread throughout the island, following the same protocol with staff having undergone the same training. The GBV care center within the hospital is located in a space designed to maintain the privacy and confidentiality while ensuring easy and unrestricted access. The care seekers are received by a trained staff member and offered first line support, referred to as LIVES (Listen, Inquire about needs and concerns, Validate, Enhance safety, Support)2, emotional support in the form of befriending, other essential services as described in the Essential Services Package8 and referred to other service providers if needed. The staff of these centers undergo in-service training on a specifically designed module in order to build their capacity and skill to provide assistance, which includes active listening and emotional support (befriending) and referral to other services. The centers constantly collaborate with other service providers such as the Women and Children’s Development Unit at the Divisional Secretariats, police, probation services, Legal Aid Commission and non-governmental organization etc9. Formal follow-up of survivors by staff of the care center is not promoted to ensure confidentiality and safety of the survivors and their children but voluntary follow-up visits and contacts through phone are encouraged very much.\n\nThe objectives of the current study were to obtain an overview of survivors of gender-based violence (GBV) who seek care, different types and consequences of GBV, their modes of referral, factors associated with GBV, characteristics of the perpetrators, health seeking behavior of the care seekers and the service provided by GBV Care Centers in two tertiary care settings.\n\n\nMethod\n\nA retrospective cross-sectional study of pooled data from two GBV care centers attached to two tertiary care hospitals situated in Colombo, Sri Lanka; Castle Street Hospital for Women (CSHW) and Kalubowila General Hospital (KGH), was carried out over a two-year period from January 2017 to December 2019. Both hospitals served an urban population while accepting referrals from other levels hospitals. The CSHW was an exclusive Women’s hospital.\n\nThe individual records maintained on all care seekers who had attended these two centers during the study period were perused. There were a total of 495 women (no men) seeking care for GBV from the two centers during the period of study, and all their records were available. There were seven records in which ≥5% of variables had missing values. These records were excluded resulting in 488 being selected for analysis. All the variables were directly reported by care seekers. However, three of the variables (the type of violence, the consequences of violence and reported stable relationships) needed additional interpretation by the health care professionals who entered the data into the records kept in the two GBV care centers. The professionals who worked in the GBV care centers had been trained on data collection and clear instructions had been given to them, regarding data collection and documentation of this data in the records maintained at the GBV care centers.\n\nFrom these records, detailed information such as the sociodemographic details, referral modalities, types of violence experienced, the consequences, factors associated with GBV, the characteristics of the perpetrators, health seeking behavior of those seeking care, and the services provided including outward referrals, were extracted by trained research assistants.\n\nIn addition, safety assessment of the care seekers was carried out using a safety score as recommended by the World Health Organization (WHO) Hand Book2 and highlighted in the Sri Lanka National Guideline for GBV care providers8. There were five questions to assess the future safety of those seeking care, with each positive response to a question being allocated a score of one; using the cumulative scores, a risk score was calculated. The following questions were asked:\n\nHas the violence increased in the past year?\n\nDoes the perpetrator consume drugs or alcohol?\n\nHas the perpetrator threatened to kill you?\n\nDoes the perpetrator keep a weapon in the house?\n\nAre you afraid to go home?\n\nAll the records were retrieved and there was no sampling involved. Therefore, there was no selection bias. Only the records which had significant (≥5%) missing values were excluded. All the research assistants were trained for data collection and they contacted the second author (RP) for any queries and clarifications. Therefore, there was uniformity in data collection, and any bias during interpretation of data was excluded.\n\nThe extracted data was entered into data collection sheets (available as Extended data)10, subsequently entered into an ongoing password-protected database and stored confidentially. Percentages and 95% confidence intervals (CIs) were calculated for the categorical variables. Possible associations of different factors with the types of GBV were assessed using chi square test, odds ratios (OR) and 95% CI. A p-value < 0.05 was considered as significant. Only grouped data are presented, ensuring confidentiality of the care seekers.\n\nApproval was obtained from the Ethical Review Committee of KGH (PL/MO/2020- application no 843). Participant consent was waived because of the anonymity of the records. Permission to carry out the study was obtained from the directors of the two hospitals KGH and CSHW.\n\n\nResults\n\nThere were 395 (80.9%) from CSHW and 93 (19%) from KGH (p<0.001). Out of 488 women in the study, 358 (73.4%) were married. There were more married women presenting for care at CSHW compared to KGH (275/395 vs 83/93, p<0.001). There were more pregnant women presenting for care at CSHW compared to KGH (170/395 vs 10/93, p<0.001). There were more teenagers presenting for care at CSHW compared to KGH (68/395 vs 4/93, p<0.001). The proportion of women ≥40 years was larger at KGH compared to CSHW (37/93 vs 49/395, p<0.001) (Table 1).\n\nKGH, Kalubowila General Hospital; CSHW, Castle Street Hospital for women.\n\naComparison between CSHW and KGH using Chi Square test.\n\nA large majority (71%) who attended these two centers had been referred from the wards and outpatient department (OPD). Of the 39 (8%) women referred by the police, 38 had been referred to KGH. The police contributed to 41% of referrals to the KGH and only 0.3% of referrals to CSHW. Other sources or referral, such as general practitioners, other hospitals and lawyers, contributed to 30.4% in KGH compared to 3.0% in CSHW (Table 2).\n\nKGH, Kalubowila General Hospital; CSHW, Castle Street Hospital for women; OPD, outpatient department.\n\nawomen who were referred from other hospitals, general practitioners and by lawyers etc.\n\nbone response was missing\n\nNearly all (94.3%) had suffered emotional violence. Economic, physical and sexual violence had been experienced by 66.6%, 64.5% and 30.3% of women, respectively, and 180 (37%) of women were pregnant at the time of violence. All four types of violence, i.e. physical, sexual, emotional and economic, were more common in those referred from the wards and OPD compared to other modes of referrals. In total, 10.8% were self-referrals and almost all of them were suffering from emotional violence. Almost all the women who had been referred by the police had experienced physical and emotional violence. All eight women referred from field health staff had emotional and economic violence (Table 3). In cases of self-referral, most women had come to know about the GBV care center through a friend. Posters displayed at these centers also contributed to their knowledge (Table 4). Most women had reported more than one type of violence and 14.5% had reported all four types. Of the 488 women, 69% had experienced both emotional and economic violence while 64% had experienced both physical and emotional violence.\n\nOPD, outpatient department.\n\naWomen who were referred from other hospitals, general practitioners and by lawyers etc.\n\nbOne response was missing.\n\ncFour responses were missing.\n\nd Seven responses were missing.\n\nesix responses were missing.\n\naAfter attending a seminar, reading a book, listening to somebody else’s story.\n\nFeeling depressed was the commonest emotional consequence and sleeping disturbances were the next commonest. Suicidal tendencies had been reported by 20%. Lack of interest in sexual relationships was the commonest sexual consequence and bruises and blackouts were the commonest physical consequences (Table 5). In 94% of women, the husband, lover or the living in partner had been the perpetrator. However, there were women who had been subjected to GBV by both husband / lover or the living in partner as well as by other members of the family (Table 6).\n\naParents, grandparents, brothers, sisters, sons, and daughters\n\nbAunts, uncles, cousins, nieces, nephews\n\nOf the 317 women who had suffered physical violence 83% had been married (OR=4.3, 95% CI 2.8-6.6, p<0.001) and 39% had been employed (OR=1.7, 95% CI 1.1-2.6, p=0.009). Of the 148 women who had suffered sexual violence, 49% had been pregnant at the time of violence (OR=2.0, 95% CI 1.4-3.0, p <0.001). Of the 180 pregnant women with GBV, 80% had suffered economic violence (OR=2.6, 95% CI 1.7-4.1, p<0.001). Although 60% of those who had suffered sexual violence were married, marriage and reported stable relationships were associated with reduced risks of sexual violence (OR=0.4, 95% CI 0.3-0.6, p<0.001, and OR=0.5, 95% CI 0.3–0.7, p=0.001, respectively). A reported stable relationship was also associated with a reduced risk of economic violence (OR=0.2, 95% CI 0.2-0.4 p<0.001 (Table 7).\n\nCI, confidence interval; OR, odds ratio.\n\naOne response was missing.\n\nbTwo responses were missing.\n\ncThree responses were missing.\n\ndFour responses were missing.\n\neFive responses were missing.\n\nfSix responses were missing.\n\ngNine responses were missing.\n\nhTen responses were missing.\n\niEleven responses were missing.\n\nOf the 488 women 90% had spoken to someone about the violence at some point of time. In 55.1% it was either the mother/mother in law or another female family member, and it was a male family member only in 20.9%. While some had confided with a friend, very few women had spoken to a public health midwife, general practitioner or the medical officer in the outpatient department (Table 8). On assessing the future safety of the women who had undergone GBV, the two leading risk factors were that the violence had increased during the previous year (80%), and that the perpetrator consumed alcohol or drugs (60%). Of the 488 women who had undergone GBV 18.6% were afraid to go home (Table 9). Out of a possible total of five, 19% of women had a score of ≥3 indicating a significant risk of repeat violence in the future (Table 10).\n\nNearly all women who attended these centers had received emotional support. Of the 488 women with GBV, 182 (37.3%) had been referred to services within the hospital and 64 (13%) had been referred to a psychiatrist. A total of 180 women (36.9%) were referred for services outside the hospital. These included in-depth counselling, and referral to social services, the police and for legal aid. Less than 20% of women were able to bring to the GBV care center a family member and the perpetrator for discussion and counseling respectively. Less than 5% of women voluntarily came for a follow-up visit (Table 11). Only two women came for a second follow-up visit.\n\naFour responses were missing.\n\n\nDiscussion\n\nThe policy of the GBV care centers is to provide services to both men and women survivors. However, all care seekers who attended these two centers were women. This may be because the majority of survivors of GBV are women11–13, and gender norms discourage men from disclosing GBV14. Furthermore, men may not be aware of the services, and one of the centers is situated in a hospital exclusively for women. The reasons for the larger numbers presenting to the GBV care center at CSHW could be because it was better known to the public, having been established two years prior to the period of study, and also because women were more comfortable and expected better care from a center situated in a women’s hospital rather than a center situated in a general hospital. The larger number of teenagers presenting with GBV to CSHW may also be due to the same reason. The majority of care seekers being between 20–39 years probably reflect the pattern of attendees to the two hospitals. The reason for the greater proportion of GBV care seekers, who were ≥40 years of age presenting to the KGH, is unclear. Although the percentage of older women (over 50 years) in the study population was only 3%, this group should not be disregarded when planning programmes to address GBV.\n\nInbound referrals between the two hospitals show two different patterns. At the CSHW contributions from the wards and the OPD were high, in contrast to KGH where the major contribution had come from the Police. Differences in the operational arrangements for medicolegal services between the two hospitals, and linkages the individual centers have developed with other service providers may account for this difference. Only one in ten women being self-referrals, in spite of the fact that the centers had been operational for several years, and messages are displayed in large bill boards at the two hospitals, is of concern. The Protocol of the GBV care center indicates the need for promoting free access to survivors without formal referrals15. It is also necessary to increase awareness among the public of their right to be not subjected to GBV, and also about the availability of GBV care centers.\n\nThe fact that 180 (37%) women attending the GBV care centers were pregnant indicates that the establishment of antenatal and postnatal screening should be considered, as recommended by the American College of Obstetrician and Gynaecologists 201216. However, the large number of pregnant women in the current study is possibly due to a sample bias because 395 out of the 488 women were from a center which is situated in an exclusively women’s hospital. By contrast, in a large community based study involving 786,464 women, carried out in 2004 in the Badulla district, physical abuse was reported in only 4.7% of a current pregnancy6.\n\nThe high emotional impact of GBV was evident, with 93% having had depression and 20% having had suicidal ideations. The proportion having suicidal ideation was higher among women who attended these centers relative to an earlier study carried out in 2007 in an agricultural community in the south-western region of Sri Lanka where domestic violence and abuse were seen as a cause of self-harm by ingestion of pesticide in 12% of the cases17.\n\nIt is important to identify women undergoing GBV and to provide effective emotional support in order to ensure their emotional wellbeing and prevent suicides.\n\nThe fact that a high risk of GBV was associated with women who were married, those who did not have stable relationships, those who were pregnant and in those who were employed, and the husband, lover or living in partner was the commonest perpetrator in both present and past episodes, indicates the vital importance of providing family counselling services to the community at large. It is important to note that 60% of perpetrators had consumed alcohol or drugs. This aspect too can be addressed by a family counselor. In the current study, however, less than 20% of women had made use of the opportunity of obtaining these counselling services from the two centers and very few women had returned to the care center for follow up.\n\nHelp-seeking by the women was mainly focused on friends and relations. This may be due to a reluctance to divulge to an outsider the fact that they had been victims of GBV. Although very few women had sought assistance from health care providers in the community, public health midwives, general practitioners and medical officers in the community are very good sources of assistance and care to survivors of GBV. The public should be educated about this.\n\nThe main strength of this study is its large sample size (n=488) and the very high data retrieval rate (95%). However, the data were collected from GBV care centers, situated in two teaching hospitals, one of which was a hospital for women and both hospitals are situated in urban areas. Furthermore, the effects of racial/ethnic differences on GBV were not studied. Therefore, the generalizability of these findings to the community at large is limited. Nevertheless, this study gives some valuable insights to the problem of GBV in two urban areas of Sri Lanka.\n\n\nConclusions\n\nEstablishment of dedicated GBV care centers within hospitals, especially those with maternity and gynaecology services, could promote identification of GBV survivors, and delivery of essential services to them. It is important to recognize the gravity of the emotional impact on survivors of GBV and provide effective and adequate emotional support to assist them and prevent suicide, when managing these survivors, especially if they are pregnant. GBV care centers need to be supported through a network of professionals skilled in family counseling to support the efforts made by the staff to help in conflicting resolution. Effective public awareness programmes on the availability of Gender Based Violence care centers dedicated to survivors of GBV and also the services provided by these centers should be conducted to ensure optimal utilization of these centers and provide care to survivors of GBV who often suffer at home in silence.\n\n\nData availability\n\nThese data are available from the Medical Directors of the KGH and CSHW, which are governed by the Ministry of Health, Sri Lanka. Restrictions apply to the availability of these data and are not publicly available. Readers or reviewers who wish to access these data and also those who are interested to use these data for future research can contact Gender and Women’s Health unit, Family Health Bureau (info@fhb.health.gov.lk), Ministry of Health, Sri Lanka.\n\nResearch data were stored in a tabular format, in SPSS (Statistical Package for the Social Sciences, Version 25). Numbers 1–93 and 94–488, contained data from the GBV care centers at KGH and CSHW respectively. Each row represents data from an individual woman and each column to represents a variable from the data sheet. There were 141 variables in the data sheet.\n\nFigshare: Gender-based violence: Experiences from two tertiary care settings in Sri Lanka. https://doi.org/10.6084/m9.figshare.12084219.v110.\n\nThis project contains the data extraction sheets used in the present study.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nSincere thanks to Professor Malik Goonewardene for investigation, statistical analysis & interpretation, and review & editing of the manuscript, Dr. Nishad AAN for providing initial statistics analysis, Dr. P. P. Nagahawatta, staff nurse N.S.Liyanage at CSHW, Dr. H. M. Karunathilake, Dr. D. P. N. Sanjeewanie, Dr. M.A. U. Perera, staff nurse Sunethra Dissanaayake at KGH for their support in data extraction. This study could not have been achieved without the dedicated support of all of them.\n\n\nReferences\n\nChristian M, Safari O, Ramazani P, et al.: Sexual and gender based violence against men in the Democratic Republic of Congo: effects on survivors, their families and the community. Med Confl Surviv. 2011; 27(4): 227–46. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: Health care for women subjected to intimate partner violence or sexual violence: A clinical handbook (No. WHO/RHR/14.26). World Health Organization. 2014. Reference Source\n\nGarcia-Moreno C, Jansen HA, Ellsberg M, et al.: Prevalence of intimate partner violence: findings from the WHO multi-country study on women's health and domestic violence. Lancet. 2006; 368(9543): 1260–9. PubMed Abstract | Publisher Full Text\n\nPels T, van Rooij FB, Distelbrink M: The Impact of Intimate Partner Violence (IPV) on Parenting by Mothers Within an Ethnically Diverse Population in the Netherlands. J Fam violence. 2015; 30(8): 1055–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSri Lanka Demographic and Health Survey 2016. Department of Census and Statistics (DCS) and Ministry of Health, Nutrition and Indigenous Medicine.2017. Reference Source\n\nMoonesinghe LN, Rajapaksa LC, Samarasinghe G: Development of a screening instrument to detect physical abuse and its use in a cohort of pregnant women in Sri Lanka. Asia Pac J Public Health. 2004; 16(2): 138–44. PubMed Abstract | Publisher Full Text\n\nChisholm CA, Bullock L, Ferguson JEJ 2nd: Intimate partner violence and pregnancy: epidemiology and impact. Am J Obstet Gynecol. 2017; 217(2): 141–144. PubMed Abstract | Publisher Full Text\n\nHealth sector response to gender-based violence- National guideline for first contact point health care providers, Sri Lanka. 2019.\n\n>World Health Organization: Country profile on gender-based violence in Sri Lanka.Reference Source\n\nPathiraja D, Pathiraja R, Senanayake L: Gender-based violence: Experiences from two tertiary care settings in Sri Lanka. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12084219.v1\n\nGuruge S, Jayasuriya-Illesinghe V, Gunawardena N, et al.: Intimate partner violence in Sri Lanka: a scoping review. Ceylon Med J. 2015; 60(4): 133–8. PubMed Abstract | Publisher Full Text\n\nVidanapathirana M: Factors related to wife-battering; a medico-legal analysis. Galle Med J. 2014; 19(1): 6–10. Publisher Full Text\n\nVadysinghe AN, Edussuriya D, Lakma MRY, et al.: Sociodemographic Profile: Nature of Abuse and Help Seeking Behavior of Victims of Intimate Partner Violence Presenting to Two Tertiary Care Units in the Central Province of Sri Lanka. Int J Med Toxicol Forensic Med. 2018; 8(1 (Winter)): 21–28. Publisher Full Text\n\nUnited Nations High Commissioner for Refugees: Working with men and boy survivors of sexual and gender-based violence in forced displacement. 2012. Reference Source\n\nProtocol for Gender-based violence care centers Mithuru Piyasa - Natpu Nilayam 2012, Family Health Bureau, Ministry of Health, Sri Lanka.\n\nAmerican College of Obstetricians and Gynecologists: ACOG Committee Opinion No. 518: Intimate partner violence. Obstet Gynecol. 2012; 119(2 Pt 1): 412–7. PubMed Abstract | Publisher Full Text\n\nKonradsen F, Hoek Wv, Peiris P: Reaching for the bottle of pesticide--a cry for help. Self-inflicted poisonings in Sri Lanka. Soc Sci Med. 2006; 62(7): 1710–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "62515",
"date": "27 Apr 2020",
"name": "Sir Sabaratnam Arulkumaran",
"expertise": [
"Reviewer Expertise Obstetrics and Women’s Health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article “Gender-based violence: Experience from two tertiary care settings in Sri Lanka” is based on the retrospective analysis of well recorded data of 488 women who attended special clinics in one women’s hospital and one general hospital in the capital city of Colombo, Sri Lanka. The analysis consists of socio-demographic factors, details of care seekers, how they ended in the clinic and the types of violence i.e. economic, emotional, physical or sexual. The tendency for suicide was notable with 20% respondents admitting that they had encountered these thoughts. In 92% of cases, the husband, partner or lover were the perpetrators. Married women and those not in a stable relationship experienced more physical violence. Emotional and economic violence were the commonest form of GBV.\nThis study highlights the important issues of economic and emotional GBV. This may be linked to the fact that these women may be no or low wage earners. The low follow up visit rates indicates that either the women are busy or they feel that the advice/services they receive may not be adequate. The conclusion suggests that there is a need to increase public awareness. One wonders whether this could be achieved by incorporating the GBV issue in schools and religious events. This paper is an important contribution to highlight the problem. Future studies are needed to focus on effective interventions and outcomes.\nThe work is clearly and adequately presented with citation to recent references. It is a detailed analysis of well recorded retrospective data. Sufficient details on methods and analysis is provided for others to produce similar work. I am not a qualified statistician to verify the accuracy of the analysis. Availability of the source data and how to access the data is provided by the authors. Conclusions are supported by the data and analysis provided.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "62514",
"date": "30 Apr 2020",
"name": "Vathsala Illesinghe",
"expertise": [
"Reviewer Expertise Violence against women"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents findings of a desk review or secondary analysis of data routinely collected at two health care institutions in Sri Lanka from those who seek services for GBV (intimate partner violence predominantly) during a specified time period.\n\nWhile this type of study can provide a descriptive summary of the characteristics of those who seek care and the nature of services that could be provided to them, it will not achieve all of the objectives nor support all of the conclusions stated in the paper. For example, the mentioned objective(s) of \"obtain(ing) an overview of [...] factors associated with GBV\" would not be possible without an analytical component/comparison group who did not experience GBV as opposed to those who did not report specific types of GBV (as in table 7).\n\nThe following aspects of this paper could be improved to increase the relevance and useful of this publication. The results section of this paper will benefit from a revision (see details below).\n\nIntroduction:\n\nThe focus of the study is not clear whether it is Gender Based Violence (GBV), GBV against women: most GBV in Sri Lanka is male violence against women in the context of marital or intimate relationships.\n\nJustify the choice of study settings: These two hospitals represent diverse health care institutions, one being a maternity hospital and a referral center for the rest of the country requires a justification as to why these two hospitals were chosen; it is relevant for the interpretation of results.\n\nMethods:\nEthics approval: Desk reviews would not require a statement about individual consent. Whether protocols/pre-approvals are in place to use routinely collected data for research purposes could be mentioned. How was anonymity of data assured? Were there any issues of data inconsistency in the two settings?\n\nSome of the questions used to assess safety were different from the WHO questions:\nHas the physical violence happened more often or gotten worse over the past 6 months? Has he ever used a weapon or threatened you with a weapon? Has he ever tried to strangle you? Do you believe he could kill you? Has he ever beaten you when you were pregnant? Is he violently and constantly jealous of you?\nWhat is the rationale for the use of different questions? The questions used by the authors could be more appropriate to the study setting; this could be justified using literature and other evidence.\n\nResults:\nThe statement 'there were 395 (80.9%) from CSHW and 93 (19%) from KGH (p<0.001)' is not meaningful without a baseline for comparison. Are you saying that there is a higher proportion of women who reported IPV among all the women/patients who were admitted to the hospital in CSHW compared to the KGH? If so, could this be because CSHW is a women's hospital and only admits women compared to KGH which admits both men and women? A proportion of 395/total cases collected would not be a meaningful comparison.\n\nInterpretation of the results should be reviewed throughout. Rather than \"there were more teenagers presenting for care at CSHW compared to KGH (68/395 vs 4/93, p<0.001)' it is better stated as 'a higher proportion of those who presented to CSHW were aged <19 years compared to the proportion of women who were of that age group presenting to KGH.\n\nTable 7. Characteristics of the women associated with the types of gender-based violence is hard to interpret without proportions. Interpretation is not clear 'of the 317 women who had suffered physical violence 83% had been married (OR=4.3, 95% CI 2.8-6.6, p<0.001)' as opposed to what? (those who did not suffer physical violence - but could they have suffered other types of violence? Which types?)\n\nTables 8 and 9, the calculation/interpretation of the confidence intervals are not clear.\n\nDiscussion:\nThe authors have addressed some of the biases and discussed salient points of this study. The results of a care center could not be compared to community based samples and prevalence.\n\nThis could be an error - \"a large community based study involving 786,464 women,\" - the sample size of this study was much smaller.\n\nThe authors must also pay attention to making conclusions about multiple variables without doing multivariate analysis - for example, it cannot be concluded that - \"a high risk of GBV was associated with women who were married, those who did not have stable relationships, those who were pregnant and in those who were employed, and the husband, lover or living in partner was the commonest perpetrator in both present and past episodes [...] because these variables were not compared with each other.\n\nFindings about help seeking, it must be noted, is based on the experiences of a sample that presented to hospital, and as such, cannot be generalized to those who have not done so. Hence it cannot give insights in to the \"problem of GBV in two urban areas of Sri Lanka\".\n\nConclusions:\nThe findings point to the nature of services needed and gaps in those provided to women who present to two large hospitals in Sri Lanka. For example, the findings can support the need for psycho-social services for survivors and the requirements for safety planning and interventions. Any other extrapolation to the community level would not be supported by the data collected and presented here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "62512",
"date": "04 May 2020",
"name": "Achini C. Jayatilleke",
"expertise": [
"Reviewer Expertise Gender Based Violence"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 provides an overview of GBV survivors presented to two urban GBV care services.\nThe article is well written, and carries important findings that will help improve GBV service provision in Sri Lankan hospitals.\n\nThe study explains the types of survivors seeking care, the different types of violence they were experiencing, the health effects of such violence, health seeking behaviors of survivors, etc.\n\nAccording to the study, a large majority had suffered emotional and economic violence, although physical or sexual violence were the reasons for referral to the centers. This finding needs to be highlighted and could be useful for future policy decisions on GBV referrals. The alarming number of suicidal tendencies indicate the need of their urgent psycho-social support along with follow up. However, it is disturbing to note that only 5% came for follow-ups, and only 2 attended a second follow up. This point needs discussion and recommendations should be made to improve GBV follow ups in the GBV care centers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "62513",
"date": "11 May 2020",
"name": "Nalika Gunawardena",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well designed study on an important topic.\n\nThe correct title of the one of the hospital settings is Colombo South Teaching Hospital and not Kalubowila General Hospital (KGH). The authors need to attend to the correction. It would have been worthwhile if the researchers could have substantiated the findings with a few qualitative inquiries among the users. The conclusions and the recommendations are relevant to improve the services.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-269
|
https://f1000research.com/articles/9-268/v1
|
17 Apr 20
|
{
"type": "Brief Report",
"title": "Inhibition of in vitro Ebola infection by anti-parasitic quinoline derivatives",
"authors": [
"Shawn Goyal",
"Beth Binnington",
"Stephen D.S. McCarthy",
"Didier Desmaële",
"Laurent Férrié",
"Bruno Figadère",
"Philippe M. Loiseau",
"Donald R. Branch",
"Shawn Goyal",
"Beth Binnington",
"Stephen D.S. McCarthy",
"Didier Desmaële",
"Laurent Férrié",
"Bruno Figadère"
],
"abstract": "There continues to be no approved drugs for the treatment of Ebola virus disease (EVD). Despite a number of candidate drugs showing limited efficacy in vitro and/or in non-human primate studies, EVD continues to plaque certain areas of Africa without any efficacious treatments yet available. Recently, we have been exploring the potential for anti-malarial drugs to inhibit an in vitro model of Ebola Zaire replication using a transcription-competent virus-like particle (trVLP) assay. We examined the efficacy of chloroquine, amodiaquine and 36 novel anti-parasite quinoline derivatives at inhibiting Ebola virus replication. Drug efficacy was tested by trVLP assay and toxicity by MTT assay. Both chloroquine and amodiaquine were effective for inhibition of Ebola virus replication without significant toxicity. The half-maximal inhibitory concentration (IC50) of chloroquine and amodiaquine to inhibit Ebola virus replication were IC50, Chl = 3.95 µM and IC50, Amo = 1.45 µM, respectively. Additionally, three novel quinoline derivatives were identified as having inhibitory activity and low toxicity for Ebola trVLP replication, with 2NH2Q being the most promising derivative, with an IC50 of 4.66 µM. Quinoline compounds offer many advantages for disease treatment in tropical climates as they are cheap to produce, easy to synthesize and chemically stable. In this report, we have demonstrated the potential of anti-parasite quinolines for further investigation for use in EVD.",
"keywords": [
"Ebola virus",
"antiparasitic drugs",
"antiviral activity"
],
"content": "Introduction\n\nSince its discovery in 1976, Ebola virus has been responsible for numerous outbreaks, with case fatalities varying from 20% to 90%1,2. From 2014 to 2016, West Africa was devastated with the largest Ebola outbreak in recorded history with six countries affected: Nigeria, Senegal, Guinea, Liberia, Mali and Sierra Leone. With no effective therapies or licensed vaccines available, little could be done to treat patients and prevent the spread of the virus3. Over a period of two years, Ebola virus was responsible for 28,646 reported infections, with 11,323 deaths1,3.\n\nSince the outbreak, a vaccine for Ebola virus (rVSV-EBOV) has been developed by the National Microbiological Laboratory in Winnipeg, Manitoba, Canada, and has been shown to be highly protective4. Undoubtedly, the rVSV-EBOV vaccine is to be a powerful defense against future Ebola outbreaks. However, it is unlikely to be sufficient to stop future outbreaks. As the natural host of Ebola virus remains unknown, eradication of Ebola reservoir populations is impossible5. Because of this, future spillover events are an inevitability6. Additionally, the unstable political environment of many affected African countries further complicates the effective administration of vaccines; as seen in the Democratic Republic of Congo during the current Ebola outbreak2,7. These factors emphasize the need for acute infection therapies.\n\nPrevious publications have focused on potential drugs predicted to have efficacy against Ebola virus disease (EVD) and included drugs already approved and widely used that could perhaps be repurposed as treatments for EVD8,9. Through a virtual screening process, Veljkovic et al.8 identified 267 US Food and Drug Administration (FDA)-approved drugs with possible inhibitory effects against Ebola virus. Among these were the antimalarial quinoline drugs amodiaquine and chloroquine. Chloroquine has been shown to be protective against Ebola virus disease in an in vivo mouse model9. Additionally, amodiaquine has been identified as having inhibitory effects in a pseudo-type entry assay of Ebola virus9. Furthermore, retrospective analysis of relative risks for patients from the 2014 to 2016 West Africa outbreak identified amodiaquine in combination with artesunate as having therapeutic effects. Data have suggested that patients infected with Ebola virus that were prescribed artesunate-amodiaquine had a significantly reduced risk of death compared to those that were prescribed artemether-lumefantrine, the latter treatment not being statistically significantly different from no treatment10. This suggests that the quinoline derivative drug class may have therapeutic use in the treatment of EVD.\n\nIn this article, the efficacy of 36 novel quinolines (Table 1) derivatives and previously approved quinoline compounds (amodiaquine and chloroquine) were examined for their ability to inhibit Ebola virus replication. Our results support previous reports that suggested that amodiaquine and chloroquine could be potential treatments for EVD. In addition, we identified additional, novel, quinolines that could be candidates for further study of their potential for inhibition of Ebola virus infection.\n\nThe column on the left indicates the percent inhibition of trVLP replication using 10 µM, and the column on the right indicates the drug toxicity as assessed using MTT viability.\n\nInhibition: HEK 293T cells were either transfected with replication machinery plasmids VP30, VP35, NP and Tim-1 (-L) or transfected with all replication machinery plasmids and Tim-1, allowing trVLP entry and replication (+L). Cells were treated with a 10µM final dose of each quinoline derivative, two hours pre-infection. Cells were infected with 100µL of viral stock diluted in 200µL of DMEM with 5% FBS. Cells were lysed, and luciferase activity was assessed 72 hours post-infection. Data represents three biological replicates. Data background corrected and displayed as a percentage of the positive control. Error shown is standard error of the mean. Viability: Drugs were added at a final dose of 10µM, 0.1% DMS. 26 hours later, media was replaced with 100µL of 0.5 mg/mL MTT in media without phenol red. After incubating for two hours at 37°C, 100 μL 10% SDS was added. MTT was fully dissolved at 37°C, then absorbance was read at 570nm. Wells containing no cells served as blanks. Cell viability was determined as percent absorbance of treatment to 0.1% DMSO control. Data shown represents four biological replicates. Data background corrected and displayed as a percentage of the no treatment control. Error shown is the standard error of the mean.\n\n\nMethods\n\nChloroquine diphosphate (Chl) was purchased from Abcam (catalog #ab142116) and amodiaquine dihydrochloride dihydrate (Amo) was purchased from Sigma-Aldrich (catalog #A2799) Artesunate (Art) was provided by the BioCIS research group at University Paris-Saclay, France. Novel quinoline derivatives: 2PentQ11,12, 2OHQ12, 2NH2Q13, 2COOHQ14, 2CNQ15,16, 2QQ17, 2Qi1518, 2Q16OH18, 2QPOH19, 2PQSel11, DD120, DD221, DD3 (Desmaële unpublished; see Extended data)22, DD4, (Desmaële unpublished; see Extended data)22, DD5 (Desmaële unpublished; see Extended data)22, MBN11120, MBN13220, MBN9120, KHD29123, KHD28823, MBN115 (Desmaële unpublished; see Extended data)22, MD823 (Desmaële unpublished; see Extended data)22, FZ4924, DD624, DD7 (unpublished; see Extended data)22, DD8 (Desmaële unpublished; see Extended data)22, FZ14224, TOF41125, TOF40125, MD20 (Desmaële unpublished; see Extended data)22, MBN8720, MBN14020, FS48 (Desmaële unpublished; see Extended data)22, AS65 (Desmaële unpublished; see Extended data)22, XF90617 and BS46011 were synthesized at University Paris-Saclay, France. Among them, 2PQsel and 2OHQ have exhibited in vitro and in vivo activities against the parasite Leishmania spp.26.\n\nHEK-293T (American Type Culture Collection; ATCC, Rockville, USA) were grown in polystyrene coated, 75 cm2 flasks (Sarstedt) in 15 mL of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS at 5% CO2 atmosphere and 37°C. Cells were harvested by washing using phosphate-buffered saline, followed by incubation with 2mL of trypsin/EDTA for 5 minutes in 5% CO2 atmosphere at 37°C.\n\nTo evaluate the efficacy of quinoline compounds of possible inhibition of Ebola virus, a replication competent mini-genome system developed by Hoenen et al.27 was adopted. Transcription and replication competent viral-like particles (trVLP) are used to model the Ebola virus lifecycle. Within the trVLPs resides, a mini-genome containing structural viral proteins (VP24, VP40, and GP1,2) with the addition of a luciferase reporter gene. To facilitate viral replication, target cells (HEK-293T) are transfected with expression plasmids containing the remaining Ebola replication machinery proteins (VP30, VP35, NP, and L) 24 hours prior to infection. This model represents the most comprehensive lifecycle model available while still being able to be conducted in a biosafety level 2 facility27–31.\n\nAs described by Hoenen et al.27, producer HEK-293T cells were seeded at 2.5 million cells/mL into 10cm Petri dishes in 10mL of DMEM containing 10% FBS at 5% CO2 atmosphere and 37°C. After 24 hours, cells were transfected with expression plasmids for the Ebola tetracistronic minigenome, virus replication machinery proteins, and bacteriophage T7 polymerase. The plasmids used have been previously described elsewhere29 and were: tetracistronic minigenome; p4cis-vRNA-Rluc, T7 polymerase; pCAGGS-T7, viral protein 30; pCAGGS-VP30, viral protein 35;pCAGGS-VP35, viral NP; pCAGGS-NP, viral polymerase L; pCAGGS-L. All transfections were carried out using the calcium phosphate method (CalPhos Mammalian Transfection Kit, Clontech Laboratories; catalog #631312). Plasmids are described in Hoenen et al.27. At 24 hours post-transfection, media was replaced with 15 mL of DMEM with 5% FBS. trVLPs containing supernatants were collected after 72 hours and stored at -80°C.\n\nTo assess the efficacy of compounds for inhibition of viral replication in vitro, 40,000 293T cells, quantified using a hemocytometer, were seeded in 400µL of DMEM containing 10% FBS. After 24 hours, seeding cells were transfected with replication machinery expression plasmids as well as the cellular Ebola virus attachment factor Tim-1 (plasmid pCAGGS-Tim1) using the CalPhos Mammalian Transfection Kit (Clontech Laboratories; catalog #631312)27. 24 hours following transfection, media was removed, followed by the addition of 300µL DMEM with 5% FBS. To this, 6µL of 100X drug stock prepared in 10% DMSO w/w was added. Two hours post-treatment, 100µL of viral stock diluted in 200µL of DMEM with 5% FBS, warmed to 37°C, was added to cells. The following day, media was replaced with 800µL of DMEM with 5% FBS. 72 hours post-infection, media was removed and cells were lysed in 200µL of 1x Renila Luciferase Assay Lysis Buffer (Renilla Luciferase Assay System, Promega; catalog #E2820). Luciferase activity in 20µL of lysate was measured using a Luminoskan Ascent microplate luminometer (Thermo Electron) after the addition of 100µL of Luciferase Assay Substrate diluted in Assay Buffer (Renilla Luciferase Assay System, Promega; catalog #E2820).\n\nCells were seeded into 96-well plates at a density to mimic day two (post-transfection) stage (30% confluent). Drugs were added to the final dose, 0.1% DMSO, and 26 hours later, media was replaced with 100 μL 0.5 mg/mL 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) in media without phenol red. After incubating for two hours at 37°C, 100μL 10% SDS was added. MTT was fully dissolved at 37°C, then absorbance was read at 570nm using a Biotech Epoch 2 spectrophotometer. Wells containing no cells served as blanks. Cell viability was determined as a percentage of absorbance of the background-corrected 0.1% DMSO treatment control.\n\nMeans were compared using the two-tailed, paired Student’s t-test (assuming equal variances) using Microsoft Excel, version 2016. A p-value ≤0.05 was considered significant. Error bars for Table 1 infection data are the range for n=2.\n\n\nResults\n\nTo assess the ability of quinolines to inhibit Ebola virus infection, we employed an established mini-genome model of Ebola replication, trVLP, allowing us to work under biosafety containment level 2 (CL 2) conditions27–31.\n\nIn our first series of tests, the ability of amodiaquine and chloroquine to inhibit in vitro infection of Ebola virus replication was tested (Figure 1). Following transfection with the required replication machinery and attachment receptor expression plasmids, HEK-293T cells were treated with the drugs at a concentration of 10 µM two hours pre-infection. Luciferase activity was measured 72 hours post-infection22. Both amodiaquine and chloroquine demonstrated significant reductions in luciferase activity, indicating inhibited viral transcription and replication32. Following these findings, dose-response experiments for amodiaquine and chloroquine were conducted. Inhibition activity was assessed at concentrations ranging from 10 µM to 0.31 µM (Figure 2A and 2B). Amodiaquine exhibited a half maximal inhibitory concentration (IC50) of 1.45 µM, while chloroquine exhibited an IC50 of 3.95 µM. Cellular toxicity for both amodiaquine and chloroquine was measured using an MTT viability assay (Figure 2A and 2B) to quantify drug toxicity along the concentration response22.\n\n293T cells were either transfected with replication machinery plasmids VP30, VP35, NP and Tim-1 (-L) or transfected with all replication machinery plasmids and Tim-1, allowing trVLP entry and replication (+L). Cells were treated with either 10µM amodiaquine or chloroquine two hours pre-infection or received no treatment. Cells were infected with 100µL of viral stock diluted in 200µL of DMEM with 5% FBS. Cells were lysed, and luciferase activity assessed 72 hours post-infection. Data shown represents four biological replicates. Data background corrected and displayed as a percentage of the positive control. Error bars shown are the standard error of the mean. Results from drug treatments were statistically compared with the (+) control group. * Denotes a p-value <0.05.\n\nCircular markers, solid line: 293T cells were either transfected with replication machinery plasmids VP30, VP35, NP and Tim-1 (-L) or transfected with all replication machinery plasmids and Tim-1, allowing trVLP entry and replication (+L). Cells were treated with either amodiaquine, chloroquine or 2NH2Q at concentrations of 10, 5, 2.5, 1.25, 0.625, or 0.31µM, two hours pre-infection. Cells were infected with 100µL of viral stock diluted in 200µL of DMEM with 5% FBS. Cells were lysed, and luciferase activity assessed 72 hours post-infection. Data shown represents four biological replicates. Data background corrected and displayed as a percentage of the positive control. Error bars shown are the standard error of the mean. Square markers, dashed line: 293T cells were seeded to mimic day two (post-transfection) stage (30% confluency). Drugs were added at a final dose of 0.47, 0.94, 1.88, 3.75, 7.5, 15, or 30 µM, 0.1% DMS. 26 hours later, media was replaced with 100µL of 0.5 mg/mL MTT in media without phenol red. After incubating for two hours at 37°C, 100 μL 10% SDS was added. MTT was fully dissolved at 37°C then absorbance was read at 570nm. Wells containing no cells served as blanks. Cell viability was determined as a percentage of the absorbance of treatment to 0.1% DMSO control. Data shown represents three biological replicates. Data background corrected and displayed as a percentage of the no treatment control. Error bars shown are the standard error of the mean.\n\nTo rapidly assess the inhibitory ability of the 36 novel quinoline derivatives (Table 1), drugs were screened at a concentration of 10 µM as described above, with drug treatments occurring two hours pre-infection. From these tests, 2OHQ12, 2NH2Q13, 2QQ17, 2Qi1518, MD823, TOF411, DD2, DD1, and MBN91 were identified as having inhibitory activity in our in vitro assay (Table 1). However, due to drug toxicity or availability of synthesized drug, only 2NH2Q was pursued for further study. A dose-response for 2NH2Q was conducted, with inhibition being assessed between 10 µM and 0.31 µM (Figure 2C). Results indicated the IC50 of 2NH2Q to be 4.66 µM, slightly higher than either amodiaquine or chloroquine.\n\n\nDiscussion\n\nAs outbreaks of EVD continue to occur, treatment and preventative strategies are urgently needed.In August 2018, the Ministry of Health of the Democratic Republic of the Congo declared a new outbreak of Ebola virus disease in North Kivu Province. As of January 19, 2019, there have been 636 confirmed cases of EVD and 370 confirmed deaths2,7. Previously, it was suggested that anti-malarial drugs may be effective for the treatment of EVD8–10. In the report, we have provided supporting evidence that antiparasitic drugs can inhibit Ebola virus replication in vitro, which could be a breakthrough in fighting EVD if proven by additional studies or clinical trials.\n\nQuinoline is a heterocyclic aromatic organic compound and is the base structure for many small molecule drugs. Quinoline-based drugs are widely used for the treatment of malarial infections. As their production is cheap, they are easy to synthesize with high yields, and are chemically stable while being readily accessible, quinolines offer many advantages for the treatment of infectious disease in tropical climates, such as the climates found in Africa8,27.\n\nOur study aimed to test anti-malarial compounds against an Ebola replication model using the previously published trVLP in vitro assay, which has been shown to accurately reflect CL4 studies using intact infectious Ebola virus29. We find amodiaquine to be effective at inhibiting Ebola viral replication. Amodiaquine is a useful anti-malarial agent in regions where Ebola virus outbreaks are possible, and this drug has been suggested to help with EVD outbreaks10. We also show using our assay that other antiparasitic malarial drugs can inhibit Ebola virus replication with low cellular toxicity. These include chloroquine, 3/35 novel synthetic quinolines, one termed 2NH2Q, with high efficacy in inhibiting Ebola virus replication and low toxicity, and 4/35 showing moderate efficacy in inhibiting Ebola virus replication while being relatively non-toxic (Table 1).\n\nQuinolines, such as amodiaquine or chloroquine, have been used in regions at risk for EVD to prevent malaria and some have stated that use of these compounds is correlated with reduced death from EVD, suggesting that quinolines could be useful as both an anti-malarial and EVD treatment/prevention drug.\n\nIn summary, 38 compounds selected for their antiparasitic activity were evaluated against Ebola virus and their cytotoxicity was also determined. Some of them had antileishmanial activity, such as 2PQsel13 and others exhibited antiplasmodial activity, such as chloroquine, amodiaquine and BS46033. The most interesting compounds emerging from viral inhibition and cytotoxicity assays were chloroquine and amodiaquine, with the advantage of chloroquine having no toxicity at 10 µM. From the quinoline series, the most promising compounds were 2Qi15 and 2NH2Q, both of them being 2-substituted quinolines. When studying structure-activity relationships, the presence of an amino group at the extremity of the propylene chain (compound 2NH2Q) allowed a better cell viability (81%) than a hydroxyl group (compound 2OHQ, 31%), despite their similar capacity for viral inhibition (89%). Replacement by a carboxylic group (compound 2COOHQ) strongly reduced the antiviral inhibition but maintained good cell viability. The other substitutions performed on the quinoline scaffold were not really satisfactory in regard to their biological activity.\n\nOur results support the hypothesis that antiparasitic quinolines active against malaria and leishmaniasis could be efficacious as a treatment for EVD. Further studies are necessary to confirm the utility of quinolines as anti- Ebola virus therapeutics, including consideration of these drugs when designing future Ebola drug trials in Africa.\n\n\nData availability\n\nHarvard Dataverse: Inhibition of in vitro Ebola infection by anti-parasite quinoline derivatives. https://doi.org/10.7910/DVN/ONGV6I22\n\nThis project contains the following underlying data:\n\n- Underlying luciferase and MTT data for Figure 1 and Figure 2 and Table 1 (in XLSX format)\n\n- Key to alternative compound names (in XLSX format)\n\nHarvard Dataverse: Inhibition of in vitro Ebola infection by anti-parasite quinoline derivatives. https://doi.org/10.7910/DVN/ONGV6I22\n\nThis project contains the following extended data:\n\n- Supplemental data.DOCX (details of the synthesis of the unpublished compounds)\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 wish to thank Mrs. B. Seon-Meniel (BioCIS) for her help in the preparations of the drugs.\n\n\nReferences\n\nWHO: WHO | Ebola virus disease. WHO. World Health Organization; 2018. Reference Source\n\nWHO: Ebola | Ebola situation reports: Democratic Republic of the Congo. Who. 2019. Reference Source\n\nColtart CEM, Lindsey B, Ghinai I, et al.: The Ebola outbreak, 2013-2016: old lessons for new epidemics. Philos Trans R Soc Lond B Biol Sci. 2017; 372(1721): pii: 20160297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMedaglini D, Siegrist CA: Immunomonitoring of human responses to the rVSV-ZEBOV Ebola vaccine. Curr Opin Virol. 2017; 23: 88–94. PubMed Abstract | Publisher Full Text\n\nRewar S, Mirdha D: Transmission of ebola virus disease: an overview. Ann Glob Heal. 2014; 80(6): 444–51. PubMed Abstract | Publisher Full Text\n\nHaydon DT, Cleaveland S, Taylor LH, et al.: Identifying reservoirs of infection: a conceptual and practical challenge. Emerg Infect Dis. 2002; 8(12): 1468–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDR Congo Ebola outbreak: Beni attack halts outreach work. BBC News. Reference Source\n\nVeljkovic V, Loiseau PM, Figadere B, et al.: Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection [version 2; peer review: 2 approved]. F1000Res. 2015; 4: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadrid PB, Chopra S, Manger ID, et al.: A systematic screen of FDA-approved drugs for inhibitors of biological threat agents. PLoS One. 2013; 8(4): e60579. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGignoux E, Azman AS, de Smet M, et al.: Effect of Artesunate–Amodiaquine on Mortality Related to Ebola Virus Disease. N Engl J Med. 2016; 374(1): 23–32. PubMed Abstract | Publisher Full Text\n\nFakhfakh MA, Franck X, Fournet A, et al.: Expeditious preparation of 2-substituted quinolines. TETRAHEDRON LETT. 2001; 42(23): 3847–3850. Publisher Full Text\n\nFakhfakh MA, Fournet A, Prina E, et al.: Synthesis and biological evaluation of substituted quinolines: Potential treatment of protozoal and retroviral co-infections. Bioorganic Med Chem. 2003; 11(23): 5013–23. PubMed Abstract | Publisher Full Text\n\nRavichandran V, Mekarnia N, Pomel S, et al.: New Water-Soluble Polymeric Prodrugs of 2-n-propylquinoline: Synthesis and Evaluation of In Vitro and In Vivo Activities Against Leishmania donovani. Regen Eng Transl Med. 2018; 4(1): 11–20. Publisher Full Text\n\nFakhfakh MA, Franck X, Fournet A, et al.: Preparation of quinolines substituted at the 2 or 3 position by an alkenyl or alkynyl chain. Synth Commun. 2002; 32(18): 2863–75. Publisher Full Text\n\nAkagah B, Lormier AT, Fournet A, et al.: Oxidation of antiparasitic 2-substituted quinolines using metalloporphyrin catalysts: Scale-up of a biomimetic reaction for metabolite production of drug candidates. Org Biomol Chem. 2008; 6(24): 4494–7. PubMed Abstract | Publisher Full Text\n\nNakayama H, Desrivot J, Bories C, et al.: In vitro and in vivo antileishmanial efficacy of a new nitrilquinoline against Leishmania donovani. Biomed Pharmacother. 2007; 61(2–3): 186–8. PubMed Abstract | Publisher Full Text\n\nGrassi F, Guimarães Corrêa AB, Mascarenhas RE, et al.: Quinoline compounds decrease in vitro spontaneous proliferation of peripheral blood mononuclear cells (PBMC) from human T-cell lymphotropic virus (HTLV) type-1-infected patients. Biomed Pharmacother. 2008; 62(7): 430–5. PubMed Abstract | Publisher Full Text\n\nSchmidt F, Champy P, Séon-Méniel B, et al.: Chemicals possessing a neurotrophin-like activity on dopaminergic neurons in primary culture. PLoS One. 2009; 4(7): e6215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFournet A, Mahieux R, Fakhfakh MA, et al.: Substituted quinolines induce inhibition of proliferation of HTLV-1 infected cells. Bioorganic Med Chem Lett. 2003; 13(5): 891–4. PubMed Abstract | Publisher Full Text\n\nNormand-Bayle M, Bénard C, Zouhiri F, et al.: New HIV-1 replication inhibitors of the styryquinoline class bearing aroyl/acyl groups at the C-7 position: Synthesis and biological activity. Bioorganic Med Chem Lett. 2005; 15(18): 4019–22. PubMed Abstract | Publisher Full Text\n\nLoiseau PM, Gupta S, Verma A, et al.: In vitro activities of new 2-substituted quinolines against Leishmania donovani. Antimicrob Agents Chemother. 2011; 55(4): 1777–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoyal S: “Inhibition of in vitro Ebola infection by anti-parasite quinoline derivatives”. Harvard Dataverse, V3, UNF: 6:LwDtfowex6sH5zxXSFP+9w== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/ONGV6I\n\nMekouar K, Mouscadet JF, Desmaële D, et al.: Styrylquinoline derivatives: A new class of potent HIV-1 integrase inhibitors that block HIV-1 replication in CEM cells. J Med Chem. 1998; 41(15): 2846–57. PubMed Abstract | Publisher Full Text\n\nZouhiri F, Mouscadet JF, Mekouar K, et al.: Structure-activity relationships and binding mode of styrylquinolines as potent inhibitors of HIV-1 integrase and replication of HIV-1 in cell culture. J Med Chem. 2000; 43(8): 1533–40. PubMed Abstract | Publisher Full Text\n\nBénard C, Zouhiri F, Normand-Bayle M, et al.: Linker-modified quinoline derivatives targeting HIV-1 integrase: Synthesis and biological activity. Bioorganic Med Chem Lett. 2004; 14(10): 2473–6. PubMed Abstract | Publisher Full Text\n\nNakayama H, Loiseau PM, Bories C, et al.: Efficacy of orally administered 2-substituted quinolines in experimental murine cutaneous and visceral leishmaniases. Antimicrob Agents Chemother. 2005; 49(12): 4950–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoenen T, Watt A, Mora A, et al.: Modeling the lifecycle of ebola virus under biosafety level 2 conditions with virus-like particles containing tetracistronic minigenomes. J Vis Exp. 2014; (91): 52381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiedenkopf N, Hoenen T: Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Viruslike Particle Assays. In: Methods Mol Biol. Humana Press Inc.; 2017; 1628: 119–31. PubMed Abstract | Publisher Full Text\n\nMcCarthy SDS, Majchrzak-Kita B, Racine T, et al.: A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus. PLoS Negl Trop Dis. 2016; 10(1): e0004364. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee N, Shum D, König A, et al.: High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system. Antiviral Res. 2018; 158: 226–37. PubMed Abstract | Publisher Full Text\n\nLe Sage V, Cinti A, McCarthy S, et al.: Ebola virus VP35 blocks stress granule assembly. Virology. 2017; 502: 73–83. PubMed Abstract | Publisher Full Text\n\nEkins S, Coffee M: FDA approved drugs as potential Ebola treatments [version 2; peer review: 2 approved]. F1000Res. 2015; 4: 48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGantier JC, Fournet A, Munos MH, et al.: The effect of some 2-substituted quinolines isolated from Galipea longiflora on Plasmodium vinckei petteri infected mice. Planta Med. 1996; 62(3): 285–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "62501",
"date": "27 May 2020",
"name": "Veljko Veljkovic",
"expertise": [
"Reviewer Expertise Bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors investigated the repurposing of anti-malarial drugs for the treatment of Ebola virus disease. The presented study of anti-malarial quinolines showed that these compounds effectively inhibit Ebola virus replication in vitro. These results point out anti-parasitic quinoline compounds as a template for the development of novel safe and cheap drugs for the treatment of the Ebola disease. This clearly presented and experimentally well-supported manuscript is suitable for indexing in F1000Research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": []
},
{
"id": "64097",
"date": "02 Jun 2020",
"name": "Jonas Šarlauskas",
"expertise": [
"Reviewer Expertise Medicinal Chemistry",
"heterocyclic compounds"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of the current report investigated an in vitro activity of 36 anti-parasitic quinoline derivatives against Ebola infection. A study represents a novel, original and important information about significant antiviral activity of selected quinoline compounds against this dangerous infection. To my mind, the paper is well written, general structure and length of the current manuscipt are fully acceptable and it is recommended to be indexed it in the present 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? 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/9-268
|
https://f1000research.com/articles/8-1439/v1
|
15 Aug 19
|
{
"type": "Research Article",
"title": "Where is the patient’s chair? Differences in general practitioner consultation room layouts - an exploratory questionnaire",
"authors": [
"Mayara Floss",
"Kyle Hoedebecke",
"Josep Vidal-Alaball",
"Mayara Floss",
"Josep Vidal-Alaball"
],
"abstract": "Background: Health clinic design varies from country to country. The layout of a general practitioner’s (GP’s) consulting room may influence the physician’s or patient’s experience. The aim of this study is to explore and investigate the layout of GP’s consulting rooms around the world and to describe any significant differences. Methods: Between 3rd July and 2nd August 2018, an internet-based questionnaire on Google Docs was distributed by email, social media and WhatsApp platforms to several worldwide rural medicine groups. Analysis of an internet-based questionnaire to explore possible layouts of consultation rooms within practices was performed. The questionnaire was designed with three distinct sections: first, a GP demographic profile including gender, year of graduation from medical school, country of graduation, and type of practice (private or public); second, questions relating to the office layout; third, a section for questionnaire feedback.\n\nResults: 502 responses to the questionnaire were received; 65.3% women and 34.7% men. The most common layout in Europe and America was where the physician and the patient were separated by a desk. The layout where the physician and the patient had a 90º angle facing each other was the most commonly used layout in Asia-Australia and Africa. For GPs who graduated before 1990 and between 1990-2010, the layout where the table was between the patient and physician was preferred. However, physicians graduating after 2010 preferred a layout with the physician and the patient with a 90º angle facing each other. Conclusion: The position of the GP’s desk differs between and within countries as well as the gender of the physician and year of graduation. Next steps should focus on gathering an even greater breadth of GP input, as well as comparing and contrasting those to the preferences of our patients and communities.",
"keywords": [
"primary health care",
"health facilities",
"facility design and construction",
"doctor-patient relations",
"health communication",
"family medicine"
],
"content": "Introduction\n\nGeneral practitioner’s (GP’s) consulting rooms differ from country to country, not only in language and cultural influences, but also in architectural design and placement of exam tables, desks, and chairs. Communication proves integral for GPs1. The incorporation of computers and electronic medical records has further changed the physical environment and the way communication occurs. A GP consultation is one of the central experiences of the patient-physician interaction with both the location of the GP’s desk and the furniture arrangement impacting that encounter2.\n\nA clinical encounter requires a doctor, a patient, and two chairs at a minimum. Additionally, most practices will also have an office desk as well as a computer. These elements (two chairs and a desk) can be placed in various arrangements. Dannenberg & Burpee argue that the design focusing solely on safety regulations, such as following building codes and avoiding fire hazards, lose all the possibilities of improving the doctor and patient experience3. Though these safety aspects are extremely important, engineers should not forego consideration of the medical encounter itself. For example, the physician’s desk can compartmentalize the physician and patient in opposite spaces - effectively serving as a “barrier” and interfering with communication4. Furthermore, the power relationship can be directly affected by the GP’s desk position, including the computer5,6 and furniture arrangement that can negatively influence the physician-patient relationship. In some cases, the introduction of the computer in the consultation room has led patients and physicians to make comparisons with the past and the “two players” consultation where computers - the third party - was not present7. In one study that included a focus group, patients preferred to sit beside the physician with a clear view of the computer screen8.\n\nThe question of where the GP should sit to promote optimal patient-centered care was raised in a Google Group discussion of the World Organization of Family Doctors (WONCA) Rural Working Party. After several email exchanges, the authors decided to develop a questionnaire-based survey. The aim of this study was to explore and investigate the layout of GP’s consulting rooms around the world and to describe any significant differences.\n\n\nMethods\n\nA specific questionnaire exploring this topic was not found and the authors decided to develop one. This was an exploratory questionnaire intended to examine the possible layouts of consultation rooms within practices.\n\nBetween July 3rd and August 2nd 2018, an internet-based questionnaire on Google Docs (Extended data) was distributed by email to several worldwide rural medicine social media and WhatsApp groups. The communities form a part of the WONCA Rural Working Party network and contain approximately 1200 GPs and trainees.\n\nThe questionnaire was designed with three distinct sections. First, the GP demographic profile was collected including gender, year of graduation from medical school, country of graduation, type of practice (private or public), if they were in a rural/remote or urban area, and if there was a computer in their practice. The second part included questions relating to the office layout (Figure 1). We proposed four scenarios as suggested in the Google group discussions. If the practice design was not represented in the scenarios proposed, the participants had the option to describe their own set up. Finally, there was a section for questionnaire feedback in order to allow for future improvements. This included a free text section and a question about satisfaction of the questionnaire (ranked from extremely dissatisfied to extremely satisfied).\n\nParticipants could opt to describe their own practice if none of the scenarios matched.\n\nThe four scenarios proposed with a computer were (Figure 1):\n\nScenario 1: GP and patient with a 90° angle facing the desk with the computer on the corner.\n\nScenario 2: GP and patient directly facing each other with the computer at the desk.\n\nScenario 3: GP and patient separated by a desk with a computer in the middle/corner\n\nScenario 4: GP and patient with a 90° angle facing each other with the computer on the corner.\n\nOther scenarios: Describe your own consulting room design\n\nWe analyzed the results of the study questionnaire using Chi-squared tests. The results were considered significant with p<0.05. The statistical programs Epi Info v7.2.2.1 and SPSS v23 were used for the statistical analyses. We did not analyze the qualitative responses in this study as they were intended to improve the design of a future questionnaire.\n\n\nResults\n\nIn total, 502 responses were collected during the 30-day period the questionnaire was active; 328 of the respondents (65.3%) were women and 174 (34.7%) were men. The country of practice with the most respondents was Romania with 199 responses (39.6%), followed by Spain with 140 (27.9%), Brazil with 51 (10.2%), Mexico with 32 (6.4%) and India with 16 (3.2%). We asked respondents to describe their practice area as rural/remote or urban; 114 respondents described their area as rural/remote (22.7%) and 379 as urban (75.5%). A total of 296 respondents described their practice as public (59%) and 184 as private (36.7%).\n\nRegarding computers at the practice, 482 out of 502 (96%) respondents had a computer at their practice and 20 (4%) did not. All except one of the respondents who didn’t have a computer were from developing countries - mainly Nigeria (40%), India (15%), and Brazil (15%).\n\nWe analyzed the countries of practice in four main geographical regions: Europe, Asia-Australia, the Americas (North and South America), and Africa.\n\nIn relation to practice layout, the most common setup in Europe was scenario 3, with the doctor and the patient separated by a desk (66%). This is also the most widely used layout in the Americas (46%), closely followed by scenario 4, with the GP and the patient with a 90° angle facing each other (41%). Scenario 4 was also the most common layout in Asia-Australia (80%) and Africa (56%).\n\nWhen comparing geographical regions, scenario 3 was used significantly more often in Europe than in the Americas (p=0.0003), whereas scenario 4 is used significantly more often (p<0.0005) in the Americas than in Europe. We did not analyze differences in the layouts between Asia-Australia and Africa as we had so few responses. Layouts of practice by geographical are reported in Table 1.\n\nThere were no statistically significant differences in the layout of the practice depending upon whether the practice was rural or urban (scenario 1, p=0.411; scenario 2, p=0.627; scenario 3, p=0.953; scenario 4, p=0.360).\n\nWe analyzed responses according to the respondents’ year of medical school graduation and established three cohorts: before 1990, between 1990 and 2010 and after 2010. Amongst those who graduated before 1990, the most common scenario was number 3 (54%), followed by scenario 2 (27%). Amongst those who graduated between 1990 and 2010, the most common scenario was number 3 (71%). Amongst those who graduated after 2010, the most common layout was scenario 4 (55%), followed by scenario 3 (31%). Scenario 2 was used significantly more often by respondents who graduated before 1990 than by respondents who graduated between 1990 and 2010 (p=0.003) and those who graduated after 2010 (p=0.003). Scenario 3 was used significantly more often by respondents who graduated between 1990 and 2010 than respondents who graduated before 1990 (p=0.0002) and those who graduated after 2010 (p<0.0005). Finally, Scenario 4 was used significantly more often by respondents who graduated after 2010 than by respondents who graduated before 1990 (p=0.0002) and respondents who graduated between 1990 and 2010 (p=0.0002). Layouts of practice by year of graduation are reported in Table 2.\n\nMost female GPs reported using scenario 3 (66%), followed by scenario 2 (16%). Male GPs also used scenario 3 most often (44%), but scenario 4 (28%) was the second most popular layout. Scenario 1 was used statistically significantly more often by male rather than female respondents (p=0.01), while scenario 3 was used statistically significantly more often by female than male respondents (p<0.0005). In contrast, scenario 4 was used more often by male than female respondents (p=0.0002). Layouts of practice by gender are reported in Table 3.\n\nFinally, the participants rated the questionnaire with 104 (20.9%) extremely satisfied, 187 (38.6%) moderately satisfied, 185 (37.1%) neither satisfied or nor dissatisfied, 16 (3.2%) moderately dissatisfied and 6 (1.2%) extremely dissatisfied. In addition, the participants’ main feedback of how to improve the questionnaire was to allow participants to design their own consultation room and/or to have picture illustrations.\n\n\nDiscussion\n\nThis study found that the position of the GP’s desk differs internationally, within countries, and according to year of graduation as well as the gender of the GP. Each approach has a social and cultural context (Figure 2).\n\nAll images were authorized for use by the general PR actioners who sent in the pictures.\n\nThe position of the chairs and desk can directly affect the doctor-patient relationship. Jacobs has previously described that arranging chairs perpendicularly (e.g. Scenario 1) implies teamwork and support, chairs placed side-by-side augments education and instruction, while chairs arranged face-to-face (e.g. Scenario 2) show expertise and can be used to gain compliance9. Scenario 3 was the setup most widely used in Europe. Because of the computer interference and the table in between, this layout does not necessarily support ‘expertise’ of the physician while simultaneously distancing the patient9.\n\nIn this study, women preferred scenario 3 (66%), followed by scenario 2 (16%). Generally, female GPs reassure and encourage patients more compared to their male counterparts1 who most often used scenario 3 (44%) - noting that scenario 4 (28%) was the second most popular. The choice of a more “protective” layout with the desk separating physician and patient could be a gender-related as GPs are increasingly facing violence, harassment, and threatening behavior10.\n\nComputers can increase the accuracy of the patient record and enhance physician efficiency but can also negatively interfere with the physician-patient interaction. The majority of GP’s (96%) in this study reported having a computer in the room. Frankel et al. listed the possible aspects in which a computer may interfere with a consultation: organization of the visit, verbal and nonverbal behavior, computer navigation and spatial navigation of the consulting room. Frankel also stated that “computering” could affect communication, which is the most powerful instrument available to the physician11. Sinsky and Beasley compared the risk of texting while driving to the risk of “texting while doctoring”; physicians should not practice “computer-centered medicine”12. Therefore, consulting rooms must be designed so that the computer is not the center of care but so that it is convenient and suited to the ergonomics of the physician. The multi-tasking involved in using a computer and the flow of the patient-physician-computer interaction can be complex and stressful11. A study conducted in Belfast examining the content of consultations with a significant psychological component showed that when the patient had a psychological complaint, physicians focused more on the patient rather than the computer and balanced their use of the computer and communication13. Moreover, it has been shown that changing the layout of a consultation room has the potential to improve communications between patients and doctors14. Clinicians who have more barriers integrating the computer in the consulting room could have more difficulties dealing with multi-tasking and this could have a negative effect on the physician-patient relationship11,15.\n\nThe patient experience is constructed consciously and unconsciously, and design affects patient perception4,9. Why is design layout in Europe different from the Americas? Why is scenario 3 preferred in Europe but scenario 4 preferred in the Americas? Although there is no definitive answer for these questions, it is interesting to reflect on the learning matrix from different countries and experiences.\n\nAlso note the dynamic reflected by the year of graduation. For those who graduated before 1990, the table between the patient and general practitioner is preferred (54%), and this increases to 70% for graduates between 1990–2010, and then decreases to 55% for younger GPs (graduating after 2010) who prefer scenario 4. This may reflect the emphasis on a more patient-centered approach the authors have noticed in recent years.\n\nIt has been suggested that consultation spaces need to be more adaptable so that additional family members can be included when necessary9 and - when using the computer - preferably the patient should be able to see the computer screen16. However, the need to understand each community and cultural competency are linked to the consulting room design. Modifying current consulting room designs should open-up new opportunities of interaction between the GP and patients.\n\nThis study has several limitations. First, the number of scenarios was limited and it was difficult to determine patient and GP locations from participants’ descriptions. This cohort of respondents represents those who have access to the Internet or knowledge of this survey - noting limited numbers of respondents from Africa, North America, and Northern Europe. The results were concentrated in some countries and continents more than others where a broader representation would likely offer better data. Furthermore, other factors such as different designs to those in the proposed scenarios, satisfaction of doctors and patients in the chosen design for the consulting room and cultural differences were not covered by this research could influence consulting room design. The studies that exist have focused mainly on the developed world experience and we were unable to expand the scope much into developing countries. Lastly, the patient’s viewpoint and preferences were not taken into consideration. Nevertheless, this study serves as an interesting starting point for further investigations to understand consulting room design worldwide and reflect upon it.\n\n\nConclusions\n\nThe position of the patient’s chair differs between and within countries in addition to distinctions based on gender and year of graduation. Modifying current consulting room designs opens up new opportunities for interaction between GPs and their patients, while continuing to remain conscious of both community and cultural differences during planning. Next steps should focus on gathering an even greater breadth of GP input as well as comparing and contrasting those to the preferences of our patients and communities.\n\n\nEthical approval\n\nThe study protocol has been approved by the Catalan Institute in Primary Care Research (IDIAP Jordi Gol) Health Care Ethics Committee (code P18/202). Prior to starting the questionnaire, participants read the following: “Dear Participant: This pilot questionnaire aims to collect data for a study about consulting room from all around the world in Family Medicine/General Practice offices. The participation and engagement in this questionnaire is free, voluntary and anonymous. Thank you for participating. -Research Team.” Participant consent was therefore obtained if the participant continued with the questionnaire.\n\nTo ensure confidentiality, all data has been anonymized.\n\n\nData availability\n\nOpen Science Framework: Where is the Doctor’s Chair, https://doi.org/10.17605/OSF.IO/9FWU717.\n\nThis project contains the following underlying data:\n\nSpreadsheet containing anonymized questionnaire responses\n\nOpen Science Framework: Where is the Doctor’s Chair, https://doi.org/10.17605/OSF.IO/9FWU717.\n\nThis project contains the following extended data:\n\nQuestionnaire\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nvan den Brink-Muinen A, Verhaak PF, Bensing JM, et al.: Communication in general practice: differences between European countries. Fam Pract. 2003; 20(4): 478–85. PubMed Abstract | Publisher Full Text\n\nAlmquist JR, Kelly C, Bromberg J, et al.: Consultation room design and the clinical encounter: the space and interaction randomized trial. HERD. 2009; 3(1): 41–78. PubMed Abstract | Publisher Full Text\n\nDannenberg AL, Burpee H: Architecture for Health Is Not Just for Healthcare Architects. HERD. 2018; 11(2): 8–12. PubMed Abstract | Publisher Full Text\n\nSheldon H, Sinayuk L, Donovan S: Designing GP buildings. Staff and patient priorities for the design of community healthcare facilities in lambeth. Oxford; 2007. Reference Source\n\nNoordman J, Verhaak P, van Beljouw I, et al.: Consulting room computers and their effect on general practitioner-patient communication. Fam Pract. 2010; 27(6): 644–51. PubMed Abstract | Publisher Full Text\n\nRouf E, Whittle J, Lu N, et al.: Computers in the exam room: differences in physician-patient interaction may be due to physician experience. J Gen Intern Med. 2007; 22(1): 43–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPearce C, Arnold M, Phillips C, et al.: The patient and the computer in the primary care consultation. J Am Med Inform Assoc. 2011; 18(2): 138–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRose D, Richter LT, Kapustin J: Patient experiences with electronic medical records: lessons learned. J Am Assoc Nurse Pract. 2014; 26(12): 674–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJacobs K: Patient Satisfaction by Design. Semin Hear. 2016; 37(4): 316–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIacobucci G: Violent crime at GP surgeries is on the rise, figures show. BMJ. 2017; 358: j4236. PubMed Abstract | Publisher Full Text\n\nFrankel R, Altschuler A, George S, et al.: Effects of exam-room computing on clinician-patient communication: a longitudinal qualitative study. J Gen Intern Med. 2005; 20(8): 677–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSinsky CA, Beasley JW: Texting while doctoring: a patient safety hazard. Ann Intern Med. 2013; 159(11): 782–3. PubMed Abstract | Publisher Full Text\n\nChan WS, Stevenson M, McGlade K: Do general practitioners change how they use the computer during consultations with a significant psychological component? Int J Med Inform. 2008; 77(8): 534–8. PubMed Abstract | Publisher Full Text\n\nAjiboye F, Dong F, Moore J, et al.: Effects of revised consultation room design on patient-physician communication. HERD. 2015; 8(2): 8–17. PubMed Abstract | Publisher Full Text\n\nKazmi Z: Effects of exam room EHR use on doctor-patient communication: A systematic literature review. Inform Prim Care. 2013; 21(1): 30–9. PubMed Abstract | Publisher Full Text\n\nAls AB: The desk-top computer as a magic box: patterns of behaviour connected with the desk-top computer; GPs' and patients' perceptions. Fam Pract. 1997; 14(1): 17–23. PubMed Abstract | Publisher Full Text\n\nHoedebecke K: Where Is the Doctor’s Chair. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/9FWU7"
}
|
[
{
"id": "55281",
"date": "15 Nov 2019",
"name": "Ferdinando Petrazzuoli",
"expertise": [
"Reviewer Expertise dementia",
"communication skills"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 addresses an interesting topic: how orientation and seating arrangements may influence the doctor patient’s relationship. The manuscript is interesting but a bit poor in terms of references. The topic has a lot to do with personal communication and I strongly advice to introduce the concepts and the references illustrated below.\n\nOrientation refers to body angles adopted when people talk, such as directly facing or shoulder to shoulder. It concerns the position of the trunk, rather the head, and it marks the degree of intimacy in the conversation and the levels of friendship.1 It is useful to look at proximity and orientation together since it has been found that there can be an inverse relationship between them that is, direct face to face alignment is linked to greater interpersonal distance and sideways angling to closer distance.1 This would be expected in situations where orientation was being used to compensate for excessive closeness.2 Orientation can also be used to include or exclude others from the group during discussion.1 Early studies of seating behaviour by Sommer3 in North America, replicated by Cook4 in the UK, point to some interesting differences in seating arrangements if individuals are given a choice of where to sit when involved in different sorts of activities. Environmental factors are also important. The physical setting can influence our mood and how we perceive the social situation. It can also determine our likelihood of interacting with others, and also influences the form that interaction will take and how long it is likely to last.1 Hall distinguished between fixed-feature and semi-fixed feature elements of the environment.5 The former includes everything that is relatively permanent or not easily modifiable, such as the architectural layout of a house, size and shape of rooms, and the material used in their construction while semifixed features are much easier to move around or modify, and include furniture, lighting, temperature. According to Knapp and Hall based on such characteristics, we form impressions of our surroundings organized around 6 dimensions.6 These are:\nFormality: concerns cues leading to decisions about how casual in what is said and done or if a more ritualized or stylized performance is demanded.\n\nWarmth: here one feels comfortable, secure, and at ease in what are regarded as convivial surroundings.\n\nPrivacy: has to do with the extent to which interactors feel that they have the space to themselves or whether others may intrude or eavesdrop.\n\nFamiliarity: involving impressions of having encountered this type of setting before and knowing how to deal with it (or not as the case maybe).\n\nConstraint: concerns perceptions of how easy it is to enter and leave the situation.\n\nDistance: address how close, either physically or psychologically, we feel to those with whom we share the space.\n\nThese perceptions shape the types of interactions we engage in and how we experience them.1 The way in which work space is arranged and utilised sends strong signals about the status and authority of occupants, the kinds of tasks and activities being implicitly proposed, and last but not least the desirability and appropriateness of focused communication in that situation.1\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "58086",
"date": "05 Feb 2020",
"name": "Miguel-Angel Mayer",
"expertise": [
"Reviewer Expertise Electronic Health Records",
"Comorbidities",
"Quality of clinical data",
"Participatory health",
"Social media"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a very interesting subject: the current general practitioner consultation room layouts in several countries. The study is based on an internet-based questionnaire.\n\nThere are some aspects that require some clarifications. For instance, the concept of \"health clinic design\" is not clear enough. It seems advisable to use another terminology such as \"consultation room design\" or \"healthcare settings design\".\n\nThe authors mention that the aim of this study was also to describe any significant differences among consulting rooms around the world; what do “significant differences” mean? In terms of? Please specify.\n\nIn the introduction, what does “the power relationship” sentence mean? It should be clarified.\n\nIn the methods section the sentence “A specific questionnaire exploring this topic was not found” is stated. How did the authors arrive to this conclusion?\n\nPlease, review this sentence: “there was a section for questionnaire feedback in order to allow for future improvements.”\n\nThere is some information missing in the methods and results sections: the sentence “distributed by email to several worldwide rural medicine social media groups” is mentioned, please specify in which social media groups or platforms they were distributed or the criteria for selecting them, the number of questions, the fact that among them 296 respondents described their practice as public and 184 as private and that means there were some respondents missing. The number of countries that participated in the study, why are Asia and Australia are included in the same group? Apart from the graduation year, the age of participants is not included, and it would have been of interest to have a more complete respondents’ profile.\n\nIn the discussion section the authors mention that “The position of the chairs and desk can directly affect the doctor-patient relationship”. This statement should be supported by several references.\n\nIn the conclusion section we read that “Modifying current consulting room designs opens up new opportunities for interaction between GPs and their patients…” What kind of modifications are the authors referring to and in which of the scenarios described? What kind of opportunities? It seems that it is of great interest to describe these points even briefly.\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": []
}
] | 1
|
https://f1000research.com/articles/8-1439
|
https://f1000research.com/articles/9-266/v1
|
17 Apr 20
|
{
"type": "Case Report",
"title": "Case Report: Colorectal cancer metastasis to a cervical lymph node – an unusual source of a neck lump",
"authors": [
"Oliver J Wright",
"Anthony Bashyam",
"Lisa Pitkin",
"Silvana Di Palma",
"Anthony Bashyam",
"Lisa Pitkin",
"Silvana Di Palma"
],
"abstract": "Colorectal cancer (CRC) is the third most common cancer worldwide, and approximately 25% of patients already have metastases at the time of diagnosis. The most common metastatic sites for CRCs are the liver, lung, bone and brain and peritoneum. Cervical lymph node metastases in CRC are rare, particularly in the absence solid organ involvement. Here we present a case of a 73-year-old female patient who, following resection of a poorly differentiated caecal adenocarcinoma, re-presented four years later with a left level IV lymph node which was ultimately found to contain metastatic adenocarcinoma.",
"keywords": [
"Colorectal cancer",
"neck lump",
"Cervical lymph node",
"metastatic caecal adenocarcinoma",
"otolaryngology"
],
"content": "Introduction\n\nCervical lymphadenopathy refers to the pathological enlargement of cervical lymph nodes. Generally, it is reactive, benign and self-limiting, but in some patients is a presenting sign of an occult malignancy.\n\nMalignant lymphadenopathy may be primary (Hodgkin lymphoma, Non-Hodgkin lymphoma) or metastatic (squamous cell carcinomas of the skin and upper aerodigestive tract or salivary/thyroid gland carcinomas). Metastatic spread is typically along lymphatic channels from the head and neck, but in 1% of cases originates more distally (most commonly from the breast, lung, kidney, tests and cervix)1.\n\nColorectal cancer accounts for 10.2% of cancer incidence worldwide, with nearly two million cases diagnosed in 2018 and makes up 9.2% (881,000) of all cancer-related deaths2.\n\nAs with many malignancies, the development of metastatic disease is associated with a poorer prognosis. Approximately 25% of patients with colorectal cancer will have metastases at the time of diagnosis, and approximately half will go on to develop them3. Colorectal malignancy can spread distally via lymphatic or haematogenous routes and typically metastasises to the liver, lung, bone, brain and peritoneum4.\n\nHere we present the case of a patient presenting with a neck lump that was ultimately found to be a metastatic caecal adenocarcinoma. The case describes an unusual pattern of disease spread, both anatomically and by histological subtype. The presence of cervical nodal disease in the absence of solid organ metastasis is particularly unusual.\n\n\nCase presentation\n\nA 73-year-old female patient presented with small bowel obstruction and underwent an emergency right hemicolectomy for a caecal mass.\n\nThe patient had multiple comorbidities: chronic obstructive pulmonary disease, hypertension, deep vein thrombosis, non-ST-elevation myocardial infection, coronary artery bypass graft, osteoarthritis and a hiatus hernia. The patient was a current smoker with a 13-pack year history and minimal alcohol consumption.\n\nPost-operative histology described a poorly differentiated caecal adenocarcinoma with clear resection margins and 4/20 regional nodes involved (TNM staging, T4N2M0). The colorectal multidisciplinary team (MDT) meeting recommended adjuvant chemotherapy to reduce the risk of cancer recurrence and treat any micro-metastatic disease. The recommendation was to complete eight cycles of capecitabine (130mg/m2) and oxaliplatin (1000 mg/m2 twice daily) chemotherapy over six months. Unfortunately, the patient only completed one cycle, declining further treatment due to the severity of side effects.\n\nAt one-year follow up, colonoscopy and a computerised tomography (CT) abdomen demonstrated no evidence of recurrent disease. Three years later, on surveillance imaging, para-aortic lymph node metastases were detected on positron emission tomography (PET) scanning. The patient underwent external beam para-aortic radiotherapy 50.4 Gy in 28 fractions, which was well tolerated, and was discharged to regular surveillance.\n\nThree years later, a rapidly enlarging painless left-sided neck lump was detected. There was no dysphagia, odynophagia, dysphonia, loss of appetite or weight loss. On examination, there was a palpable small level IV neck lump which was smooth, non-fluctuant and non-tethered to underlying structures.\n\nRoutine blood tests, including full blood count, liver function and renal function, were unremarkable. Carcinoembryonic antigen, a tumour marker for colorectal carcinoma, was elevated at 24.0 mg/L (normal range <2.5 mg/L).\n\nUltrasound of the lump showed a 6-mm level IV cervical lymph node with intranodal calcifications, loss of hilar architecture and increased vascularity suspicious for malignancy.\n\nCytology from fine-needle aspiration of the node under ultrasound guidance showed clusters of atypical cells with pleomorphic nuclei and a moderate amount of cytoplasm. Immunohistochemical analysis of the cell block preparation showed cells which were positive for cytokeratin AE1/AE3 and CDX2. AE1/AE3 is an antibody-based assay which reacts with epithelial based tissues, and CDX2 is an intestine-specific transcription factor that is highly sensitive and specific for adenocarcinomas of intestinal origin5.\n\nWhole-body PET-CT showed elevated tracer uptake in an 8.4mm left level IV LN, and in a second 7-mm left supraclavicular lymph node with no further evidence of thoracic or abdominal recurrent disease (See Figure 1).\n\nIn the post-cancer-treatment surveillance context of this patient, any new, persistent lymphadenopathy should stimulate a high degree of diagnostic suspicion and investigations should be suitably intensive.\n\nIn the general population, the considered differential is broader. A ‘Surgical Sieve’ approach is useful in diagnosing a neck lump. The authors find the mnemonic VITAMIN-C particularly helpful (see Table 1).\n\nFollowing a thorough history and examination, the principal method of investigating a neck lump is through ultrasound imaging and if suspicious, a fine-needle aspiration or core biopsy.\n\nReactive lymph nodes have a different ultrasound appearance to malignant ones which are typically larger and more spherical in shape. An irregular border indicating extracapsular spread, hypo-echogenicity, macrocalcifications, intranodal necrosis and peripheral vascularity are all pathological features concerning for malignant disease6. Further imaging with CT, PET-CT, or MRI is useful in defining the lesion, staging disease and in radiotherapy planning.\n\nOn day 35 following re-presentation, the patient was discussed at the Head and Neck Cancer MDT in conjunction with the Colorectal Cancer MDT, the recommendation was made for a selective neck dissection of levels 2, 3, 4, and 5. The surgery went without complication, and the patient was discharged the following day.\n\nHistology from the dissection showed 6/20 lymph nodes with metastatic deposits of poorly differentiated adenocarcinoma. There was with evidence of intracellular mucous and positive staining for CK20 and CDX-2. Immunohistochemical analysis confirmed an intestinal origin (Figure 2).\n\n(a) Atypical cells with pleomorphic nuclei demonstrated Hematoxylin and Eosin stain. (b) Immunohistochemistry for CK20 with characteristic cytoplasmic and nuclear staining. (c) Immunohistochemistry for CDX-2. (d) Intracellular mucus detected on special stain (ABPAS) confirming glandular (adenocarcinoma). ABPAS is an Alcian Blue stain which demonstrates intra and extracellular mucous.\n\nThe patient recovered well from the neck dissection, and at two-month review, the patient was disease-free and asymptomatic.\n\nAt two-year review, the patient had developed some intermittent abdominal discomfort. CT imaging revealed small volume retroperitoneal and pelvic lymphadenopathy. Since she had previously received radiotherapy to the area, the oncology team felt that the only treatment they would recommend would be chemotherapy; however, the patient declined because of side-effects and her comorbidities. She remains under three-monthly surveillance in the outpatient department.\n\n\nDiscussion\n\nPathological cervical lymphadenopathy is most commonly due to metastasis from malignancies that originate in the head and neck. Common primary sites include skin cancers, salivary gland tumours, thyroid carcinomas and squamous cell carcinomas of the upper aerodigestive tract1.\n\nGenerally, malignancies metastasise along predictable routes of lymphatic drainage to nodal areas, which has led to the development of neck levels (I-VII) that guide surgical management. Investigations can be targeted to the likely primary site based on the location of the lymphadenopathy; for example, level I nodes drain the oral cavity, submandibular gland, lip and anterior nasal cavity.\n\nThe presentation of metastasis from more distant sites, particularly infraclavicular, is more challenging to account for as there is often no apparent lymphatic route. A potential mechanism of spread is via obstruction of the thoracic duct.\n\nThe thoracic duct is the largest lymphatic vessel in the body. It extends from the cisterna chyli at the level of the second lumbar vertebrae and ascends through the abdomen and thorax to terminate into the left subclavian vein. It receives afferents from the thorax, abdomen and pelvis, and therefore provides a potential route for the spread of disease to left supraclavicular lymph node. Virchow’s node is the eponymous name given to this node, and Trosier’s sign refers to it pathological enlargement, typically due to gastric or pulmonary malignancy7. Lymphatic drainage of the head and the neck is via the superficial and deep lymphatic vessels which converge to form the right and left jugular lymphatic trunks. Vessels from the right terminate in the right lymphatic duct and those on the left, in the thoracic duct. Lymphatic obstruction at supraclavicular or lower cervical nodes can, therefore, result in the retrograde spread of abdominal or thoracic disease to higher cervical or even contralateral lymph nodes1.\n\nDifferent histological subtypes of colorectal cancer also appear to have predilections for distinct metastatic sites. In an autopsy study of 1675 patients with metastatic disease, Hugen et al.8 found that adenocarcinomas preferentially metastasised to the liver, while mucinous and signet ring cell carcinomas were more frequently associated with peritoneal metastasis. Signet ring cell carcinomas metastasised to distant lymph nodes more frequently than adenocarcinomas and mucinous carcinomas. Interestingly, there appeared to be no significant difference in the rate of pulmonary metastasis between histological subtypes8.\n\nThe study also demonstrated a difference in metastases distribution based on the original tumour site. While both primarily metastasised to the liver, colonic malignancies were more likely to present with intra-abdominal metastasis (peritoneal, omental or ovarian) and rectal cancers were more frequent in extra-abdominal sites (lung, brain). The venous drainage of the rectum can explain its more wandering metastatic spread. While the superior and inferior mesenteric veins drain the colon and upper two-thirds of the rectum into the portal venous system; the lower third of the rectum is drained by the middle and inferior rectal veins which bypass the liver and directly enter the inferior vena cava. Our case is therefore made more unusual by the fact it was neither rectal or a signet ring cell carcinoma; so distant cervical metastasis is unexpected. In patients with only one distant metastasis, the site of metastases also appears to be an important prognostic factor. For example, thoracic metastases are associated with longer median survival than peritoneal, bone or nervous system metastases9.\n\nAn understanding of the different patterns of metastatic spread between subtypes is of clinical importance as it may affect both immediate management and follow up techniques. In line with guidance from the National Institute for Health and Care Excellence, patients routinely undergo at least two CT scans of the chest, abdomen pelvis in the first three years after treatment10. Given the findings of Hugen et al., a case could be made for imaging additional sites or using other imaging modalities such as PET-CT in higher-risk subtypes such as signet ring cell or mucinous carcinoma.\n\nSeveral case reports have been published on the metastatic spread of colorectal carcinoma to the thyroid gland11–13, but isolated metastatic spread to cervical lymph nodes is rare, and treatment decisions are made on a case by case basis. Aksel et al.4 reported on a case of cervical lymphadenopathy as the presenting feature of undiagnosed sigmoid colon carcinoma. The patient was treated with simultaneous resection of the colonic primary and left neck node dissection. Postoperatively the patient received adjuvant folinic acid/fluoronacil/oxaliplatin chemotherapy and was disease-free at nine-month follow up4. Ochi et al.14 reported on a case of metastatic recurrent sigmoid colon cancer to Virchow’s lymph node. This was managed non surgically with systemic chemotherapy and radiotherapy, which achieved a complete response and no recurrence at the ten-month follow-up14.\n\nIn conclusion, although most metastatic lymphadenopathy originates from head and neck primaries, clinicians should always consider the possibility of more remote disease and not underestimate the importance of a detailed past medical history and systems review. Management of metastatic disease to the neck should be discussed by the relevant cancer MDTs and may involve surgical resection and chemotherapy with or without radiotherapy.\n\n\nLearning points/take home messages\n\n1. Most metastatic neck nodes arise from primary tumours of the head and neck\n\n2. Occasionally the metastases are from intra-abdominal malignancies, most commonly breast, lung, kidney and rarely bowel.\n\n3. A thorough history and examination is essential in assessing a patient with a neck lump\n\n4. Management of metastatic disease to the neck is on a case by case basis in conjunction with the Multi-Disciplinary Team discussion\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "References\n\nLópez F, Rodrigo JP, Silver CE, et al.: Cervical lymph node metastases from remote primary tumor sites. Head Neck. 2016; 38(Suppl 1): E2374–E2385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO Factsheet. 2018; Accessed: 29/9/2019. Reference Source\n\nVatandoust S, Price TJ, Karapetis CS: Colorectal cancer: Metastases to a single organ. World J Gastroenterol. 2015; 21(41): 11767–11776. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAksel B, Dogan L, Karaman N, et al.: Cervical Lymphadenopathy as the First Presentation of Sigmoid Colon Cancer Case Report. Middle East Journal of Cancer. 2013; 4(4): 185–188. Reference Source\n\nWerling RW, Yaziji H, Bacchi CE, et al.: CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 2003; 27(3): 303–310. PubMed Abstract | Publisher Full Text\n\nAhuja AT, Ying M, Ho SY, et al.: Ultrasound of malignant cervical lymph nodes. Cancer Imaging. 2008; 8(1): 48–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZdilla MJ, Aldawood AM, Plata A, et al.: Troisier sign and Virchow node: the anatomy and pathology of pulmonary adenocarcinoma metastasis to a supraclavicular lymph node. Autopsy Case Rep. 2019; 9(1): e2018053. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHugen N, van de Velde CJ, de Wilt JH, et al.: Metastatic pattern in colorectal cancer is strongly influenced by histological subtype. Ann Oncol. 2014; 25(3): 651–657. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiihimaki M, Hemminki A, Sundquist J, et al.: Patterns of metastasis in colon and rectal cancer. Sci Rep. 2016; 6: 29765. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNICE: Colorectal cancer: diagnosis and management Clinical guideline [CG131]. Published date: November 2011; Last updated: December 2014; Accessed: 29/9/2019. Reference Source\n\nCoelho MI, Albano MN, Costa Almeida CE, et al.: Colon cancer metastasis to the thyroid gland: A case report. Int J Surg Case Rep. 2017; 37: 221–224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkimaru K, Onda M, Tajiri T, et al.: Colonic adenocarcinoma metastatic to the thyroid: report of a case. Surg Today. 2002; 32(2): 151–4. PubMed Abstract | Publisher Full Text\n\nMinami S, Inoue K, Irie J, et al.: Metastasis of colon cancer to the thyroid and cervical lymph nodes: a case report. Surg Case Rep. 2016; 2(1): 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhchi T, Akagi Y, Kinugasa T, et al.: Virchow lymph node metastatic recurrence of sigmoid colon cancer with severe lymph node metastases successfully treated using systemic chemotherapy combined with radiotherapy. Anticancer Res. 2013; 33(7): 2935–40. PubMed Abstract"
}
|
[
{
"id": "62875",
"date": "04 May 2020",
"name": "Alfio Ferlito",
"expertise": [
"Reviewer Expertise Head and neck surgery/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 manuscript is accepted in its current form. This is an interesting and well written paper. The radiological and histological documentation is adequately represented in support of the case report. Metastatic cancer in the cervical region is always a challenge to the head and neck specialist, in particular if the primary tumour site is unknown. This paper can be useful especially to a young readership of head and neck surgeons/trainees.\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": "62874",
"date": "26 May 2020",
"name": "Carlos Manuel Costa Almeida",
"expertise": [
"Reviewer Expertise General 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\nGlobally, I think this paper can be indexed, but I have to make a few remarks on some aspects that I would like the authors to address in the paper before its indexed.\nWhen the patient had the primary colon tumour, how was the CEA? It is not registered then, we only know that it was elevated when the cervical mass was noted, three years later.\n\nWhat about colonoscopy? Was it done any time during the three years of follow-up? Or not? And in a cervical mass, apparently a metastatic lymph node, in a patient who had a colon cancer three years before, shouldn’t it be done as part of the patient study?\n\nHow was the follow-up of the primary tumour done? And how should it be done? This would be a valuable piece of information for this case and its report.\n\nA cervical lymphatic metastasis in the left supraclavicular notch can originate virtually anywhere below the diaphragm, in the abdominal cavity and the testis, following the thoracic duct, as it is said. Besides the head and neck, and the left thorax, of course. The Virchow’s node and the Troisier’s sign are very appropriately referred to in the text. Such a metastasis coming from the colon or the rectum is not common, but it is not “unexpected”…\nI am sure we agree that the colo-rectal cancer prognosis improved a great deal combining an appropriate surgical treatment with an efficacious chemotherapy. It was a pity the patient did not accept chemotherapy twice.\nAnyway, I think this is the report of an interesting case, that should be indexed, giving further information on supraclavicular metastization.\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": []
},
{
"id": "74782",
"date": "27 Jan 2021",
"name": "Thomas Geyton",
"expertise": [
"Reviewer Expertise ENT"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis ia a well written interesting article which describes the presentation of a 73 year old patient with a neck lump. Key features of the medical history, imaging and histology are explored and the case described the rare event of cervical metastasis to a cervical lymph node. The authors explore the anatomical background to this presentation and contextualise in relation to the current literature.\n\nI found this article interesting, scientifically sound and coherent. I therefore have no reservations in regard to its indexing in its current form.\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
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https://f1000research.com/articles/9-266
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https://f1000research.com/articles/8-2040/v1
|
02 Dec 19
|
{
"type": "Research Article",
"title": "Study of interaction energies between residues of the active site of Hsp90 and geldanamycin analogues using quantum mechanics/molecular mechanics methods",
"authors": [
"Ricardo Vivas-Reyes",
"Alejando Morales-Bayuelo",
"Carlos Gueto",
"Juan C. Drosos",
"Johana Márquez Lázaro",
"Rosa Baldiris",
"Maicol Ahumedo",
"Catalina Vivas-Gomez",
"Dilia Aparicio",
"Alejando Morales-Bayuelo",
"Carlos Gueto",
"Juan C. Drosos",
"Johana Márquez Lázaro",
"Rosa Baldiris",
"Maicol Ahumedo",
"Catalina Vivas-Gomez",
"Dilia Aparicio"
],
"abstract": "Background: Heat shock protein (Hsp90KDa) is a molecular chaperone involved in the process of cellular oncogenesis, hence its importance as a therapeutic target in clinical trials. Geldanamycin is an inhibitor of Hsp90 chaperone activity, which binds to the ATP binding site in the N-terminal domain of Hsp90. However, geldanamycin has shown hepatotoxic damage in clinical trials; for this reason, its use is not recommended. Taking advantage that geldanamycin binds successfully to Hsp90, many efforts have focused on the search for similar analogues, which have the same or better biological response and reduce the side effects of its predecessor; 17-AAG and 17-DMAG are examples of these analogues. Methods: In order to know the chemical factors influencing the growth or decay of the biological activity of geldanamycin analogues, different computational techniques such as docking, 3DQSAR and quantum similarity were used. Moreover, the study quantified the interaction energy between amino acids residues of active side and geldanamycin analogues, through hybrid methodologies and density functional theory (DFT) indexes. Results: The evaluation of interaction energies showed that the interaction with Lys58 residue is essential for the union of the analogues to the active site of Hsp90, and improves its biological activity. This union is formed through a substituent on C-11 of the geldanamycin macrocycle. A small and attractor group was found as the main steric and electrostatic characteristic that substituents on C11 need in order to interact with Lys 58; behavior was observed with hydroxy and methoxy series of geldanamycin analogues, under study. Conclusion: These outcomes were supported with quantum similarity and reactivity indices calculations using DFT in order to understand the non-covalent stabilization in the active site of these compounds.",
"keywords": [
"Hsp90",
"geldanamycin analogues",
"PM6",
"3D-QSAR",
"QM/MM approach",
"Molecular Quantum Similarity."
],
"content": "Introduction\n\nIn recent years, molecular chaperones have been of great interest to the scientific community, since these compounds play an important role in apoptosis and cellular oncogenesis. In addition, they maintain the correct folding and three-dimensional conformation of proteins in the cell and control the balance between the synthesis and degradation of many proteins1. Such chaperones include the heat shock protein of 90KDa (Hsp90), which is ATP dependent2–6. Hsp90 are chaperones that are highly conserved in many species7–9. Under normal conditions in the cell, Hsp90 are present between 1–2% and can be found in the cytosol, nucleoplasm, endoplasmic reticulum and mitochondria10,11. Moreover, these chaperones are involved in the maturation of oncogenes and play an important role in the survival, invasion, proliferation, metastasis and angiogenesis of cancer cells12. The expression of Hsp90 is associated with many types of tumors, including breast cancer, pancreatic carcinoma, leukemia, systemic lupus, as well as resistance to many drugs13. The viability of Hsp90 as a therapeutic target in cancer is defined by: (1) its participation in folding and stabilization of a wide range of proteins involved in oncogenesis and malignant progression, which is important for maintenance of cancer cells; (2) the micro-environmental conditions found in tumor hypoxia such as low pH and a bad nutritional status, which tend to destabilize proteins, being necessary for chaperone activity; and (3) an increase in Hsp90 concentration in cancer cells14\n\nStructurally, Hsp90 have three domains, which are important for its chaperone activity, which are: (1) the N-terminal domain, highly conserved (25 KDa) whose function is ATP union; (2) the middle domain, which is the site of union of “client proteins”; and (3) the c-terminal domain, responsible for the homodimerization of theHsp9015. Thus, Hsp90 chaperone activity can be inhibited when some of these sites are blocked. In fact, Hsp90 inhibitors have been categorized according to their mode of action: (1) blocking of the binding site of ATP, (2) breakdown of the interactions of the co-chaperone/Hsp90, (3) antagonism of the union between the client protein/Hsp90, and (4) interference with the modifications post-transactional of Hsp9014. In this way, Hsp90 chaperone activity inhibition is a promising target for development of anticancer drugs13,16. One of these inhibitors is geldanamycin, an antitumor antibiotic that has the ability to join the binding site of ATP in the N-terminal of the Hsp90 domain, triggering the loss of chaperone Hsp907,14,17. However, geldanamycin shows a high cytotoxicity testing power, as well as hepatotoxicity in clinical trials, hence its disuse8,14,18. Recently, some analogues of geldanamycin have been synthesized, which showed less toxic side effects and better anticancer activity. Among these analogues, we found: 17-allylamino-17-demethoxygeldanamicyn (17AAG) and 17-desmethoxy-17-N, N dimethyl amino ethyl amino geldanamycin (17DMAG)19,20. Thus, this research focused on the search of chemical parameters that can help design the best geldanamycin analogues, which could make the toxic effects of its predecessor disappear. This paper presents an analysis based on a combination of different techniques and computational methods (docking, docking-fp6, 3DQSAR semi-empirical and residue-ligand interactions) and quantum similarity, with the purpose of shedding some light on the role of the changes affecting the activity of geldanamycin analogues when modifying substituents of C-11 and C-17 of the macrocycle position.\n\n\nMethods\n\nStudy setting. Experimental activity data was taken from the work of Tian et al.21. In this paper, the synthesis of 48 geldanamycin analogues was reported. However, only 43 analogues were considered, because only these analogues had IC50 values reported. In Figure 1, geldanamycin’s structure as well as the position of carbon atoms C-11 and C-17s are shown. These positions are responsible for the difference between analogues. In Table 1, the substituents of C11 and C17 of each analogue are tabulated with its respective biological activity expressed as pIC50.\n\nThe carbon atom C-11 and C-17s of the macrocycle of the geldanamycin are numerically indicated.\n\nDesign of structures. For the design of the structures of the geldanamycin analogues, a template of the analog 17-DMAG from protein Data bank was obtained (code: 1OSF P; Figure 2). This template has a bioactive conformation (Figure 2b), which was used to design the structures under study. For this, the substituents on C-11 and C17 of each analogue were incorporated using the design tool of Sybyl 7.3 (Tripos International)22. Molden is an alternative software for design of structures, which can be downloaded for free.\n\na) R3 indicates the position that takes the substituents on the C-11 position of the macrocycle for the analogues of series 7 and b) Structure bioactive of the 17-DMAG.\n\nOptimization of structures. Once the design of the structures for the geldanamycin analogues was completed, short molecular dynamics were performed. The macrocycle in all analogues were left rigid, as this did not lose its bioactive configuration (Figure 3). The dynamics conditions were MM3 force field, 350 K, 5 ps of heating and 30ps of duration. Then, the conformations with lower energy were selected and carried out to partial optimization, using semi empirical approach PM6 method implemented to MOPAC 200923.\n\nDocking. The optimized structures of the geldanamycin analogues were taken to perform docking calculations. The process of docking was carried out for each of the analogues. Docking was implemented in the AutoDock program 1.5.4 version and its ADT 4.2 graphic interface24. The parameters selected to perform the docking were completely flexible ligands, grid dimensions 50×50×50 Å with 0.375 Å spacing and generation of 100 poses for each ligand.\n\nOnce the results of the docking were obtained, the hydrogens were added to each of the poses obtained from AutoDock and then its optimization was carried out using PM6 semi-empirical method (AutoDock -PM6 hybrid methodology) implemented in MOPAC 2009. This procedure was carried out in order to recover the information represented in the interactions with other hydrogen atoms, which were not considered by AutoDock. This calculation also allowed information related to interaction energies at a more formal level of theory to be obtained. For the optimization with the semi-empiric method, amino acids residues of the active site within a radius of 5Å with center in the ligand was considered.\n\nBy the selection of the best poses of each methodology (AutoDock and AutoDock -PM6 hybrid methodology) the following parameters were considered: a) the pose reported by AutoDock with lower energy within the histogram of greater population and b) the best pose given by methodology hybrid (AutoDock-PM6) were selected according to the stability of complex (low energy).\n\nSubsequently, three models of 3DQSAR were obtained from: a) simple alignment based on the superposition of structures optimized analogues of geldanamycin (A model), b) alignment based on the superposition of structures obtained from docking (model B), and c) alignment based on the superposition of structures obtained from the AutoDock PM6 hybrid methodology (model C). Sybyl 7.3 program were used by alignments. The CoMFA approach was performed using PLS (Partial Least Squares) to a maximum of 10 components.\n\nIn contrast, the interaction energy of analogues-residue (I.E) was calculated to observe the behavior the geldanamycin analogues once these bound to the active site of HSP 90. Moreover, EI was used to explain the influence of interactions residues-analogues in the biological activities.\n\nFor this, the complex between analogues and residues of active site (5 Å) of HSP90 were defragmented using Sybyl and PM6 semi-empirical method (AutoDock -PM6 hybrid methodology) combination. The interaction energy of analogues-residue (I.E) was calculated:\n\nInteraction energy of analogue -residue (I.E) = EAM - EL-EM\n\nwhere EAM a EL and EM, denote analogue-residue energy, ligand energy and residue energy, respectively.\n\nThe density functional theory (DFT) calculations were used to analyze the non-covalent stability of the compounds on the active site. TGSA algorithm was used to perform these calculations25a. Quantum similarity and reactivity indices were used. A molecular quantum similarity measure (MQSM) between two systems A and B was performed. ZAB is a comparison between two molecules that can be constructed using their respective density functions (DFs). DFs can be multiplied and integrated over all the respective electronic coordinates, in turn weighed by a defined positive operator Ω(r1,r2)25b–27\n\n\n\nThe nature of the operator used in Equation 1 will provide information that will be compared between the two systems and at the same time will name our measure of similarity; when the operator chosen is the Dirac delta function (a function that finds a very useful approach for functions with high peaks, such as electronic density, and constitutes the same type of mathematical abstraction such as the charge or the point mass). This is Ω(r1,r2)= δ(r1- r2), and we obtain an overlapping MQSM, one of the first similarity measures used; another widely handled possibility is the use of the Coulomb operator, that is Ω(r1,r2)= |r1- r2|-1, obtaining a coulombic MQSM. A measure of similarity can be applied between two molecular systems, including the case in which the two molecules are equal, in this case the measurement is called measure of self-similarity (ZAA for the case of molecule A)26.\n\nGiven a group of N molecules, we can obtain, for each of them, a measure of similarity with respect to each of the group's molecules, including itself. From all these obtained measurements we can construct a symmetric matrix. The i-th column of the matrix can be considered as the collection of all measures of similarity between the i-th molecule and each element of the group, including itself. This is why each vector (each column of the matrix) can be considered as a discrete N-dimensional representation of the i-th structure. These collections of vectors can be considered as a group of molecular descriptors. However, this collection of columns of the similarity matrix does not simply constitute another group of molecular descriptors like those that are generally used to theoretically describe a given molecule; each descriptor has the following particular characteristics26,27:\n\ni Universality, in the sense that it can be obtained from any group of molecules and for any molecule within the group.\n\nii Impartiality, since in the construction process there are no other options than those provided by the knowledge of the density functions and the similarity measures involved.\n\nManipulation of MQSM and visualization techniques: Similarity indexes. Once we have chosen a group of study objects and the operator related to the MQSM in Equation 1, the measure of similarity obtained for the group is unique; however, it is common practice to transform or combine these measures to obtain a new class of auxiliary terms that can be called quantum similarity indices (QSI). There is a vast amount of possible manipulations of MQSM that lead to a variety of QSI definitions. The most used are the following and by this reason are used in this work28–30:\n\nCarbó's similarity index between two molecules I and J\n\n\n\nThis corresponds to the cosine of the angle subtended by the DFs involved, taken as vectors, for which this index is also called the cosine-like similarity index. This Carbo QSI, for any pair of molecules compared, it has a value between 0 and 1, which depends on the similarity associated with the two molecules (when I = J the index will approach 1)28–30.\n\nQuantum similarity using Euclidean distance\n\nConsidering the similarity Equation 3,\n\n\n\nfor which, if k = x = 2, it is reduced to the so-called Euclidean distance index. We can also define the index 3 of the form:\n\n\n\nThis Equation 4 constitutes the distance index of infinite order31.\n\nMolecular Quantum Similarity Measure (MQSM) definition used in this work. The measures of quantum similarity are based on psychological perception and the obvious principle of similarity: “The more similar the two molecules are to one another, the more they are similar in their properties”. From this fact, we are able to obtain a quantitative measure of quantum similarity of the range of similarity between two molecules; they are based on the comparison of their densities. For the range of similarity between two compared systems. Generally, the MQSM is a measure between two tentative density functions involving molecular systems. The density functions are multiplied and integrated for the electronic coordinates in the convenient domain. MQSM between two systems A and B, denoted ZAB, is a comparison between two molecules that can be constructed using their respective Density Functions (DFs). Both DFs can be multiplied and integrated over all the respective electronic coordinates, in turn weighed by a defined positive operator Ω(r1,r2). TGSA software was used to carry out similarity measure25a. The MSQM can be single defined at the scale of the first order molecular density functions associated with the compared molecules, and with positively defined operators26–29:\n\n\n\nwhere A and B are the two quantum objects studied, {r1, r2} are the set of electronic coordinates associated with the corresponding wave function, {ρA, ρB} of the first order and Ω (r1, r2) positively defined supported in the operator, dependent on the coordinates of the electrons24–29.\n\nTypes of measures in molecular quantum similarity. It depends, essentially, on the information required, seriously on the selection of the supported operators, producing different types of MSQM. For these calculations the TGSA program was used25a.\n\nMQSM overlap considering Equation 2\n\nThe simplest and most intuitive usual choice of a positively defined operator is the distribution Dirac's delta, Ω (r1, r2) = δ (r1, r2). This selection transforms the general definition of MQSM, specifically, to calculate the overlap MQSM, which obtains measurements of the volume enclosed in the superposition of both electronic density functions26–29:\n\n\n\nThe Dirac delta function comes intuitively from physical definition and is computationally compliant. The MQSM comes from information on the concentration of electrons in the molecule and indicates the degree of overlap between the molecular comparison25b–30.\n\nMQSM Coulomb considering Equation 2\n\nIf the operator (Ω) is adopted by the Coulomb operator, Ω (r1, r2)=1|r1−r2|, it provides the coulomb MQS, which represents the electrostatic repellent coulomb energy between two charge densities30,31:\n\n\n\nThe coulomb operator performs the effect for the overlap density functions. Considering the functions of molecular density as an electron distribution in space, this expression is only for the extension of Coulomb for the distribution of continuous charge, and for that reason it can be considered, in some occasions, as descriptors of electrostatic potential. This operator obtains the measurement of electrostatic repulsion between electronic distributions and is associated with electrostatic interactions25b–29.\n\nEuclidean distance index considering Equation 3\n\nThis is another typical transformation that can be defined according to the classical distance:\n\n\n\nwhere Δxj = xaj – xbj is the distance between the objects a and b, and k=2 for the definition of distance. The Euclidean distance between two quantum objects A and B is defined by 25b–29:\n\n\n\nOccasionally it is expressed as: DAB=ZAA+ZBB+ZZAB DAB has values in the range of [0,∞﴿ but, converges for previous cases, it has a value of zero between the compared objects, if the compared objects are identical25b–29:\n\n\n\nGeometrically this index can be interpreted by the norm of the differences between the density functions of the compared objects. The index of the Euclidean distance can be defined by the distance or dissimilarity index; the index can also be expressed as25b–29:\n\n\n\nAlignment method: Topo-geometrical superposition algorithm (TGSA). In this work, the alignment was carried out using the TGSA32 method. The TGSA was proposed by Gironés and programmed and implemented by the same author. This method considers that the optimal alignment of molecules is carried out through superposition on the common skeleton, taking only into account the type of atoms and the bond of the interatomic interactions, which is the atomic number of the coordination. Was carry out, its purpose the algorithm examines the atomic pairs of the molecules and aligns the common substructure for a series of molecules32. The method is only based on topology and geometric considerations, where the molecular topology is manifested in the way of comparing the distant bonds. In two molecules, the superposition is unique and does not depend on the type of operator chosen to provide the meaning of the similarity32.\n\nFirst, molecular coordination and atomic number are necessary to indicate the performance of the program. The molecular coordination is ordered in bases, according to the decrease of the atomic number, in order to determine a path for the number of hydrogens in the molecule32.\n\nConsidering that the superposition of hydrogens is not significant, and with the required computational requirements, the hydrogen atoms are not included in the process. The next step is the definition of the atomic pair, the duo is defined only if the pair of atoms in the box is involved, with their respective determinants, the duo has to be defined for each molecule, all molecules behave with each other with their respective meanings of interatomic distances, obtaining translocations. The translocations are taken within the fluctuations of the spine of the conformations produced by the presence of the different substitutions in the molecules32. This procedure always discards bonds that are not common with skeletons. Once the duos are compared, the algorithm creates atomic triads by adding three atoms selected from the duos. These supplementary atoms must be in the box to be compared later. In geometric terms, this generates a triangle in the plane, where the atoms occupy the vertices of the triangle, and the sides correspond to the effectiveness of the chemical box32.\n\nThe triangle obtained by a molecule is compared with the triangle obtained by the second molecule with the respective interatomic distances, and with the translational distances in the comparison duo. If the three distances of both triangles compared are similar, both triads are similarly considered and stored. The triads that do not meet the classification criteria are automatically discarded to complete the comparison, the selected triad is superimposed and the result of the molecular alignment is determined univocally32.\n\nThis process is repeated for the atoms and the algorithms chosen are those of the alignment that maximizes the number of atoms superimposed, minimizing the index CIJ, it is used by the comparison criterion of the interatomic distances and this cost calculation with the absolute value of each difference with the composite after-location:\n\n\n\nwhere dIJ:∑i:1nA∑j:1nB|xi,I−xj,J|2, n is the number of atoms and x the molecular coordination, CIJ is determined in the interval [0,1], evaluating the quantification of the overlap. This indicates better the alignment when CIJ it approaches unity, originating the ideal case of structural identity CIJ: 1.\n\nThe TGSA method considers the molecules as rigid bodies, so there is no flexibility in the structure (nothing of rotation and vibration in the distances of the angles in the box). This is designated by the operator in the homogeneous set of molecules and does not yield good results with different molecular structures; this comes from the alignment pair that is restricted with the common recognition skeleton. In contrast, this common recognition of substructures produces a coherent alignment with chemical intuition. TGSA-Flex program was used to perform this procedure because is simple and has low computational requirements32–36.\n\nSome of the present authors have shown the relationship between quantum similarity and chemical reactivity descriptors in several works37–45. In addition, the quantum similarity and DFT use the DF as an object of study. The similarity indexes, specifically the Coulomb index, can be related to electronic factors associated with chemical reactivity. All the calculations were carried out using the free software TGSA-Flex32\n\nUsing the Frontier Molecular Orbitals (FMO) and the energy gap, the global reactivity indices, such as chemical potential (μ)45–47, hardness (η)45–47 and electrophilicity (ω)45–47, will be calculated. These chemical reactivity indices give an idea about the stability of the systems.\n\nThe chemical potential (μ) characterizes the tendency of the electrons to escape from the equilibrium system, whereas the chemical hardness (η) is a measure of the resistance of a chemical species to change its electronic configuration46,47.\n\n\n\nand\n\n\n\nElectrophilicity index can be interpreted as a measure of the stabilization energy of the system when it is saturated by electrons from the external environment and is mathematically defined as39–43:\n\n\n\nIn this work, the local reactivity descriptor are the Fukui functions (Equation 16 and Equation 17, f). The Equation (16) and Equation (17) represents the response of the chemical potential of a system to changes in the external potential. It is defined as the derivative of the electronic density with respect to the number of electrons at constant external potential:\n\n\n\n\n\nwhere (fk+) is for nucleophilic attack and (fk−) for electrophilic attack44–46. In this sense, using the global and local reactivity descriptors it is possible to study the quantum dissimilarity along the molecular set.\n\n\nResults and discussion\n\nTable 2 shows the results obtained for the prediction descriptors (q2) and correlation (R2) of the three models.\n\nFrom the three models that were evaluated, only C model provided the best prediction values (q2) to all components evaluated. However, the change in q2 to 2, 5 and 7 components (48.3, 46.8 and 49.0 %, respectively) was not significant, which infers that C model was more reproducible than A and B models, as observed in Table 2.\n\nIn addition, the results obtained from B and C models provide information about receptor ligand-interactions, which can be observed in the alignments of these models (Figure 4 and Figure 5); where the conformational changes observed in analogues structures can be evidence of what may be happening at the cellular level. However, the C model is superior to B model because all hydrogens atoms were considered in receptor-ligand coupling (complex), while, B model only polar hydrogen atoms. Thus, model C is more real in terms of the interactions of analogues with the active site of HSP90 (Quantum mechanical calculations), thus providing a new alternative for docking studies7,48. The results obtained to A model were lowest; and this fact can be associated to alignment used to generate 3DQSAR, where the structures used by alignment were only optimized without considered the interaction with active site. Thus, the only difference was observed in the aligned structures were on C-11 and C-17; which are positions where geldanamycin analogues are structural different (Table 2).\n\nThe model C (CoMFA) shows the contribution of the steric and electrostatic fields which generated R2= 0.963 and q2= 0.50 for an optimal number of 7 components. On the other hand, the steric and electrostatic individual contributions were 57.6 and 42.4%, respectively. Steric and electrostatic contour maps are shown in Figure 6.\n\n(a) Steric map and (b) electrostatic map on positions C-11 and C-17 (CoMFA).\n\nIn Figure 6a and 6b, steric and electrostatic contours are depicted. These contours are located in a special way on positions C-11 and C-17 macrocycle, this is expected because these are the positions where the structural differences of the geldanamycin analogues occur.\n\nThe steric CoMFA map (Figure 6a) covers the sterically favorable contours (80% contribution) corresponding to the regions in the space where the steric volume leads to an increase in activity (green contour map), while the sterically non favorable regions (20% contribution) correspond to areas in the space where the steric volume is expected a decrease of activity. Map of green steric contour on the carbon atom C-17 position of the macrocycle indicates analogues with bulky substituents in this position tending to favor the activity, as it is reflected in the 3e ligands (pIC50 = 7.96) and 1f (pIC50 = 7.77) that have this peculiarity on carbon 11. The yellow outline indicates that the presence of bulky groups adversely, affected activity, as noted in the 4f ligands (pIC50 = 5.77) and 8i (pIC50 = 5.57). This analysis of steric maps allows us to infer that the ligands of series 1 and 3 are favored, while to the ligands of series 8 – 7 (Table 1 and Table 2) are disadvantaged.\n\nOn the electrostatic map (Figure 6b), the blue contours (80% contribution) indicate the regions in the space where the groups with low electron density favor activity, while the red contours (20% contribution) indicate regions where groups with high electron density decrease activity.\n\nThe blue contours on the C-17 position of the macrocycle indicate that the activity is favored for those ligands with substituent electroatractors in that position (ligands 1e pIC50 = 7. 62 and 3 h pIC50 = 7. 22). In contrast, the red and blue contours on the C-11 position of the macrocycle indicate that both the electronegative and electropositive substituents can promote activity provided that they should be oriented towards the contours on the map. This counts the vast majority of the ligands in this study that can present any of the two conditions set out for the substituents of C-11. General examples of this behavior are the ligands 1g (pIC50 = 7. 15) and 3d (pIC50 = 7. 62) oriented towards the blue contour. 4b (pIC50 = 6. 99), 3a (pIC50 = 7. 08), 3b (pIC50 = 6. 55) are oriented towards the red contours. It can be seen that the steric map (CoMFA) is the best explanation of the relation between structure-activity of the geldanamycin analogues, since the contours obtained from those analogues are more specific than the electrostatic ones, and give a clearer indication of the type of substituents that should be on positions C-11 and C-17 of the macrocycle.\n\nTo confirm the outcomes of this, we carried out other calculations in order to explain the behavior of this set of molecules\n\nWhile the results of the 3DQSAR were clear, they did not fulfill expectations, because they cannot explain, in all cases, the behavior between structure-activity. For this reason, the energy of interaction (I.E) residue-ligand was calculated. In order to observe the activity within the active site of Hsp90, once this was bonded to the geldanamycin analogues an accurate analysis of the interactions ligand (substituents)-receptor could be obtained. In Figure 7, the interaction energies obtained for geldanamycin have been plotted.\n\nRed arrows show the energy interaction values of Lys58, Asp93 and Lys112 residues.\n\nIn Figure 7, it is shown that the most significant values of I.E are present in Lys58, Asp93, Lys112 residues, where Lys58 is the most favorable of them, which suggests that this could possibly be a \"critical\" residue to analyze. On the other hand, it is expected that the interaction with the residue Lys58 is highly favorable because of the ease that has this residue forms a bridge of hydrogen with oxygen of the hydroxyl of the C-11 position of the macrocycle group.\n\nIn Figure 8, the interactions between geldanamycin and the active site of Hsp90 is depicted, and it can be observed that the possible \"critical\" residues can interact positively with geldanamycin, hence why the I.E may have been significant. Moreover, Lys58 can donate a bridge of hydrogen with methoxy group (C-17) and hydroxy group (C-11), but the interaction is the most favored for hydroxyl group, because the methoxy group is far from the active site26. The above does not rule out that the C-17 methoxy group does not affect the energy of the interaction with this residue.\n\nGreen lines indicate the residues donator of hydrogen bridge.\n\nIn Figure 9, interaction energies for the composite 2 (pIC50= 8.04) and 3e (pIC50= 7.96) have been plotted. 4b (pIC50 = 6.96), 6d (pIC50= 7), 8i (pIC50= 5.6) and 8h (pIC50= 5.57) present high, medium and low activities, respectively, like their predecessor.\n\nThe choice of these analogues was made in order to show the changes that have the values of I.E possible “critical\" residue when making cellular modifications on C-11 and C-17. This could corroborate the outcomes obtained which are similar to geldanamycin and Lys58, Asp93 and Lys112 residues. In addition, the significant values of I.E with Asp54, which can be considered as another possible \"critical\" residue. Interaction with Asp54 is justified by the high electron density that this residue has, which allows it to act as an acceptor of the hydrogen bridge of the substituents on C-17, which are potential donors, for instance, the substituents possessing an amine or diamine on that position (Table 1 and Table 2)\n\nIn Figure 9, it was also noted that Lys58 and Asp54 present a significant gap in values of I.E with respect to the Lys112 and the Asp93, which in turn means that interactions with these geldanamycin analogue residues are of high relevance. Interestingly, it was also found that those residues are located on positions C-11 and C-17 of the macrocycle, where precisely the substitutions occur (see Figure 8). To verify the importance of residues Lys58, Asp93, Lys112 and Asp54 as \"critical” residues, it was necessary to examine the analogues of geldanamycin by family, because the structural differences are minimal.\n\nContinuing with the analysis of interaction energy, we considered series 11-hydroxy and 11-methoxy together because these only differ in substituent on C-11. In Figure 10 and Figure 11, the values of I.E for 11-hydroxy and 11-methoxy of the geldanamycin analogues have been plotted. In these figures, it can be observed that most of the series of compounds tend to have significant I.E values with Lys58, Asp93 and Lys112 and Asp54 residues, which makes us suppose that those residues are \"critical\" for both series.\n\nThe highest values of I.E were found in Lys58 and the Asp54 (Figure 10 and Figure 11). The I.E for the Lys58 values tend to be higher and homogenous for 11-hydroxy than for 11-methoxy series, since the Lys58 forms a hydrogen bridge with oxygen more easily (the macrocycle position C-11). Besides, this group does not have major steric or repulsive impediments. In the case of 11-methoxy series, the interaction is disadvantaged because of the steric impediment that presents with the methyl group. But despite this, in both series the atom of oxygen, by effects of polarization of bond, holds a partial negative charge, which in turn makes it a good bridge acceptor of hydrogen (effect observed in all analogues of both series). However, the I.E. values for Asp54 are more fluctuating for both series since some analogues of these series cannot donate a hydrogen bridge to the residue, as is the case of the analogues 1d and 3d that lack an atom of hydrogen in the substituent at C-17 (Table 1). Hence, the low values of I.E with this residue (-7.6577 and -8.70502, respectively), which in turn leads to interactions with Gly 137, Gly97, Ile96, and Met98 residues (1b analogue). The above may be occurring because the analogues of 11-methoxy series tend to rearrange within the active site in a way that can interact with Lys58, which in turn implies it will strengthen interaction with other residues.\n\nThis behavior allows to the assumption that interaction between Lys58 and substituent on C-11 are important for biological activities of analogues with hydroxy and methoxy groups on C-11. Moreover, these analogues have the same substituent on C-17 and thus the same interaction with Asp54.\n\nIn Figure 12 the values of I.E for 11-O-acyl geldanamycin analogues have been plotted, which show the trend seen in the two previous series, since most of these compounds loses the interactions with Gly97 and Ile96 residues, and the values of I.E for Asp54 residue are similar to the Asp93. However, the trend of significant values of I.E for Lys58 as observed in previous cases is the same. The behavior observed in this series can be associated to the steric impediment of the substituents on C-11 and C-17, which impedes the formation of hydrogen bridges with Lys58 and Asp54 residues. On the other hand, as explained above, the steric impediment in these molecules induced a change, which involves the regrouping of these residues on the active site, and thus, these have to stabilize through the formation of new interactions with other residues such as Gly97 and Ile96. Also in Figure 12, it can be observed that for analogues of this series, despite having bulky groups in C-11, the interaction with Lys58 remains the highest, which suggests that the interaction with this residue has a high relevance to these analogues, and that to increase the interaction decreases the interaction with Asp54. It is of note that the analogues that cannot make a bridge of hydrogen to Asp54 have a low I.E value, as was observed with 4e and 4d analogues, which possess a methoxy group on C-17. Moreover, the non-formation of a hydrogen bond creates repulsion between oxygen atom and carboxyl group of this residue. In general, this series highlights that Lys58 acts as a \"critical\" residue and that to some extent it can affect the activity of these compounds7,49.\n\nIn Figure 13 the values of I.E for 11-ketone analogues of geldanamycin have been plotted. It can be observed that the I.E values with Lys58 are not significant as in the previous cases because Lys112, which happens to be the residue with the best I.E value, displaces these. This behavior can be explained due to the carbonyl content, which easily accepts the bridge of hydrogen donated by the Lys58 as would be expected. The preferences that have these analogues by the Lys112 residue can occur because of the short distance between carbonyl group and this residue. Besides, the rigidity that presents the double bond of the carbonyl group on C11 shields hydrogen formation with Lys58 and thus the molecule suffers a rearrangement within the active site, leading it to seek other interactions enabling it to stabilize. This was observed with Met98, Phe138 and Lys112, which had the best iteration energy.\n\nFigure 13 also shows the values of I.E for the Asp54 residue. The behavior observed was similar to that observed with the other series, but increases the interaction with Asp54 that made the interaction with Lys58 decrease. In general, with analogues of this series it can be deduced that the formation of a weak interaction with Lys58 forces the molecule to strengthen its interaction with other residues of the active site, in order to stabilize. As was previously mentioned these groups of compounds differ from other geldanamycin analogues since these only have substitution in the hydrogen of the amine located in C11 (Figure 2a) since the substituent at C-17 is the same for all analogues of this series (see Table 2).\n\nFigure 14 plots the values of I.E for 11-amine analogues and Lys112, which were more significant than those found for Lys58. This is expected because the substituents on C-11 are not the most propitious to create interaction with Lys58, because these do not possess an atom which can accept a hydrogen bond. Moreover, the volume of these substituents (Table 2), induce a reorganization in the interactions with the active site, as was observed with Lys112, Asp93, Thr184 and Asp54 residues. The latter has special mention because, as seen in Figure 15, its values of I.E are the highest, which in turn allows us to infer that in the absence of a good interaction with Lys58, the compounds in this series are stabilized in particular to strengthen its interaction with Asp54 and subsequent to this with Lys112, Asp93 and Thr184. Further interactions with Asp54 are more favored, since the substituent on C-17, can donate a hydrogen bond to the residue, when the substituent is voluminous.\n\nIn general, the fall of values of I.E for Lys58 could be due to the low activity of these compounds, which is complemented by the considerable volume of the substituents on C-11. On the other hand, the interaction these analogues have with Asp54 serves to give stability to the complexes, especially for those analogues of geldanamycin possessing a secondary amine on C-17. Figure 15 also plots the values of I.E for 11-oxime analogues of geldanamycin.\n\nThe I.E values for Lys112 are higher than Lys58 (Figure 15), with the exception of 8a where its behavior is opposite (Lys58: -17,3721 vs Lys112: -15,6787). This could be due the fact that 8a has an atom of oxygen on C-11, which can accept, without any steric problems, a hydrogen bridge with Lys58. In addition, the substituent on C-17 is the least bulky of this series, which favors their interaction. 8b and 8d possess the same substituent on C-11, so would be expected to have the same values for I.E with Lys58 as 8a (-18.8069 and -14.561, respectively), and thus these were higher than with Lys112 (-19.6651 and -18.2331, respectively). However, this behavior is not observed for the similar substituents on C-17, where these induce a change that involves strengthening the interactions with other residues to compensate the decrease of interaction with Lys58. This behavior was particularly observed in the interactions with Lys112 where this was higher than Lys58.\n\nFor the rest of the compounds of this series (8e-8i), the I.E values are to be expected to be low for Lys58 because the substituents at C-11 are voluminous analogues (Table 1), which prevents the formation of the hydrogen bridge by steric effects with Lys58, since the molecule has to be rearranged within the active site in such a way that this interaction is what gives it a certain degree of destabilization to the molecules and gives the force to strengthen interactions with other residues (Lys112). The above is supplemented by the fact that the substituents at C-17 are also bulky.\n\nIn general, the interaction between substituents on C-11 and Lys58 could have an impact on geldanamycin analog activity. However, the interaction between C-17 and Asp54 also could have an important role in activity, since substituent volume on C-11 can affect C-17 because this molecule gives priority to certain points in its site active.\n\nThese results are compared to I.E value for geldanamycin and the most active analogue 2 (Figure 16). It can be observed that I.E value for residues in both compounds do not have significant differences, which may explain the increase in the activity of 7.39 (GDM) to 8.04 (2). Thus, it can be expected that the activities of these compounds are similar or failing that, the activity of compound 2 is slightly superior to the GDM. The above tells us it would be worth reviewing the value of the biological activity of this ligand.\n\nTo understand the 3D-CoMFA outcomes, a quantum similarity study on the most reactive compounds was performed. Taking into account the reference compound GDM, in Table 3 are shown the overlap similarity indices using Equation 12. These indices allow us to make some considerations about the quality of the superposition on the molecular set. The compound with the higher structural similarity with the reference compound GDM is 1a (0,9837, Table 3) with a Euclidean distance of 1,0662 (see Table 5). Therefore, the group (-NH2) in compound 1a shows a light dissimilarity in the alignment method. This result agrees with the low steric effect to this group.\n\nThe compound with lowest similarity with respect to GDM is compound 3e (0,6489, see Table 3) with a Euclidean distance of 5,1217 (see Table 4). Compound 3e has two substituents groups with high steric effects according to Table 1. The bulky substituents can show some problems on the superposition process. Compound 2 has higher biology activity (pIC50=8.04, see Table 1) and has an overlap index with respect to the reference compound 0,9631 with an Euclidean distance of 1,6289. These compounds have two methoxy groups that are electron-donating groups. These electronic considerations have strong influence in the superposition process due to the fact that flexibility and free movement on ramifications decrease.\n\nThe main intention of this study was to observe the electronic effects of the group substitutes and Coulomb indices. Table 4 shows the values obtained with the most reactive compounds with respect to the reference compound GMD. Table 5 depicts the electronic similarity indices using the Coulomb similarity to analyze the electronic effects on group substitutes.\n\nOwing to inductive effects, the basicity of the amine group might be expected to increase with the number of alkyl groups on the amine. However, correlations are complicated owing to the effects of solvation, which are opposite to the trends for inductive effects, according to what is shown in the 3D-CoMFA maps. However, solvation effects also dominate the basicity of aromatic amines. For these compounds, the lone pair of electrons on nitrogen delocalizes into the ring, resulting in decreased basicity. Substituents on the aromatic ring, and their positions relative to the amine group also affect basicity, as seen in Table 1. To understand these electronic features, Table 6 shows the global chemical reactivity descriptors to the most reactive compounds.\n\nThe compound with higher global reactivity descriptors is 1c with chemical potential (-4,9587 eV), hardness (5,6360 eV), softness (0,1774 eV)-1 and electrophilicity (2,1814 eV). The reference compound GMD has chemical potential (-4,8982 eV), hardness (5,5895 eV), softness (0,1789 eV)1 and electrophilicity (2,1462 eV). The compound with higher biology activity 2 has chemical potential (-4,5953 eV), hardness (5,2243 eV), softness (0,1914 eV)-1 and electrophilicity (2,0210 eV) (see, Table 7). The high hardness and electrophilicity values in these compounds may be related with the non-covalent stabilization on the active site. Electrophilicity is related with the system saturation when received electrons come from the external environment. To analyses these reactivity details, the Frontier molecule is shown in Figure 16–Figure 18, and show orbitals HOMO, LUMO and the Fukui Functions 〈fk−〉≈|HOMO|2 and 〈fk+〉≈|LUMO|2, to compounds 1a, GMD (reference compound) and 2.\n\nCompound 1a has the higher structural and electronic similarity with GDM (reference compound) and compound 2 has the higher biological activity.\n\nCompound GDM show that the Fukui Functions are in the same zone, and these reactivity characteristics are very important, since these can be related with nucleophilic character on the active site and non-covalent stabilization (see Figure 19).\n\nThe reactivity contours in compound 2 are very similar to compound 1a. Therefore, these compounds have different regions to electrophilic and nucleophilic attacks. Therefore, we can see the different effects that can occur in the central ring when different substituents such as activators or deactivators are used.\n\n\nConclusion\n\nWith the data provided by the energy of interaction, we suggest that interactions that influence the activity of the geldanamycin analogues are those formed with residues such as Lys58, Lys112 and Asp54, which we refer to as \"critical\" residues. This is supported by the fact that the substituents are bulky at position C-11 and the macrocycle does not favor the interaction with Lys58, and the substituents with amines secondary in C-17 from the macrocycle position favor the interaction with Asp54.\n\nSubsequently the energy profile of each group of geldanamycin analogues showed that more active compounds have high energy of interaction with Lys58 and are less active with Lys112, regardless of the substituents onC-17. Consequently, the increase in I.E of Lys112 or Asp54 generates increased interaction energy with other residues and decrease the values of I.E with Lys58\n\n\nData availability\n\nFigshare: Receptor_Alim_2019_Pose_E_D_1.mdb (1).zip, https://doi.org/10.6084/m9.figshare.10255739.v150.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nR.V-R. is indebted to Cartagena University for continuous support to the group.\n\n\nReferences\n\nWorkman P: Pharmacogenomics in cancer drug discovery and development: inhibitors of the Hsp90 molecular chaperone. Cancer Detect Prev. 2002; 26(6): 405–410. PubMed Abstract\n\nScheibel T, Buchner J: The Hsp90 complex--a super-chaperone machine as a novel drug target. Biochem Pharmacol. 1998; 56(6): 675–682. PubMed Abstract | Publisher Full Text\n\nSmith DF, Whitesell L, Katsanis E: Molecular Chaperones: Biology and Prospects for Pharmacological Intervention. Pharmacol Rev. 1998; 50(4): 493–513. PubMed Abstract\n\nNeckers L, Schulte TW, Mimnaugh E: Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity. Invest New Drug. 1999; 17(4): 361–373. PubMed Abstract | Publisher Full Text\n\nMaloney A, Workman P: HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther. 2002; 2(1): 3–24. 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Publisher Full Text\n\nCarbó-Dorca R, Emili Besalú: A postulate involving quantum mechanical momentum in position space, density function expression of the kinetic energy and Heisenberg’s uncertainty relation. J Math Chem. 2014; 52(7): 1949–1967. Publisher Full Text\n\nAmat L, Besalú E, Carbó-Dorca R, et al.: Identification of active molecular sites using quantum-self-similarity measures. J Chem Inf Comput Sci. 2001; 41(4): 978–91. PubMed Abstract | Publisher Full Text\n\nMorales-Bayuelo A, Ayazo H, Vivas-Reyes R: Three-dimensional quantitative structure-activity relationship CoMSIA/CoMFA and LeapFrog studies on novel series of bicyclo [4.1.0] heptanes derivatives as melanin-concentrating hormone receptor R1 antagonists. Eur J Med Chem. 2010; 45(10): 4509–22. PubMed Abstract | Publisher Full Text\n\nMorales-Bayuelo A, Torres J, Vivas-Reyes R: Hückel treatment of pyrrole and pentalene as a function of cyclopentadienyl using local quantum similarity index (LQSI) and the topo-geometrical superposition approach (TGSA). Int J Quant Chem. 2012; 112: 2637.\n\nMorales-Bayuelo A, Torres J, Baldiris R, et al.: Scale Alpha and Beta of Quantitative Convergence and Chemical Reactivity Analysis in Dual Cholinesterase/Monoamine Oxidase Inhibitors for the Alzheimer Disease Treatment Using Density Functional Theory (DFT). Int J Quant Chem. 2012; 112: 2681.\n\nMorales-Bayuelo A, Torres J, Vivas-Reyes R: Theoretical Calculations and Modeling for the Molecular Polarization of Furan and Thiophene under the Action of an Electric Field Using Quantum Similarity. J Theo Comp Chem. 2012; 11: 223.\n\nMorales-Bayuelo A, Vivas-Reyes R: Topological model to quantify the global reactivity indexes as local in Diels-Alder reactions, using density function theory (DFT) and local quantum similarity (LQS). J Math Chem. 2013; 51(1): 125–143. Publisher Full Text\n\nMorales-Bayuelo A, Vivas-Reyes R: Theoretical model for the polarization molecular and Hückel treatment of PhosphoCyclopentadiene in an external electric field: Hirschfeld study. J Math Chem. 2013; 51(7): 1835–1852. Publisher Full Text\n\nMorales-Bayuelo A, Baldiris R, Torres JE, et al.: Theoretical study of the chemical reactivity and molecular quantum similarity in a series of derivatives of 2-adamantyl-thiazolidine-4-one using density functional theory and the topo-geometrical superposition approach. Int J Quantum Chem. 2012; 112(14): 2681–2687. Publisher Full Text\n\nVivas-Reyes R, Arias A, Vandenbussche J, et al.: Quantum similarity of isosteres coordinate versus momentum space and influence of alignment. Journal of Molecular Structure: THEOCHEM 2010; 943(1–3): 183–188. Publisher Full Text\n\nMorales-Bayuelo A, Valdiris V, Vivas-Reyes R: Mathematical analysis of a series of 4-acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines: a simple way to relate quantum similarity to local chemical reactivity using the Gaussian orbitals localized theory. Journal of Theoretical Chemistry. 2014; 2014: 13. Publisher Full Text\n\nVivas-Reyes R, De Proft F, Biesemans M, et al.: A DFT Study of Tin-and Crown-Ether-Based Host Molecules Capable of Binding Anions and Cations Simultaneously. Eur J Inorg Chem. 2003; 7: 1315–1324. Publisher Full Text\n\nCarbó-Dorca R, Besalú E: EMP as a similarity measure: a geometric point of view. J Math Chem. 2013; 51(1): 382–389. Publisher Full Text\n\nStebbins CE, Russo AA, Schneider C, et al.: Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell. 1997; 89(2): 239–250. PubMed Abstract | Publisher Full Text\n\nCarbó-Dorca R: Collective Euclidian distances and quantum similarity. J Math Chem. 2013; 51(1): 338–353.\n\nVivas-Reyes R: Receptor_Alim_2019_Pose_E_D_1.mdb (1).zip. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10255739.v1"
}
|
[
{
"id": "58072",
"date": "30 Jan 2020",
"name": "Jaime Enrique Plazas Román",
"expertise": [
"Reviewer Expertise bioinformatics",
"biological computation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe importance of this article lies in the active site of Hsp90 which is involved in the process of cellular oncogenesis; Geldanamycin is currently being used, however due to the hepatotoxic damage showed in clinical trials by Geldanamycin, it is necessary to look for Geldanamycin analogs. This article finds or gives solutions to this question by giving as a result through docking, 3DQSAR and quantum similarity of these analogs, so it is an interesting and outstanding result on clinical research, excellent work.\nI think this paper is excellent and is an important addition to the literature. The evaluation of interaction energies from the interaction with Lys58 residue is essential for the union of the analogues to the active site of Hsp90, and improves its biological activity. This union is formed through a substituent on C-11 of the geldanamycin macrocycle. A small and attractor group was found as the main steric and electrostatic characteristic that substituents on C11 need in order to interact with Lys 58; behavior was observed with hydroxy and methoxy series of geldanamycin analogues, under 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": "5373",
"date": "16 Apr 2020",
"name": "Ricardo Vivas-Reyes",
"role": "Author Response",
"response": "Thank you very much for your comments"
}
]
},
{
"id": "58209",
"date": "20 Feb 2020",
"name": "Leonardo C. Pacheco-Londoño",
"expertise": [
"Reviewer Expertise My area of expertise related to the article is theoretical calculations to explain points that through experimentation are not possible to explain or complementing experimental data. The area in which I have worked the most is the design of sensors using vibrational spectroscopy",
"other areas where I unwind are the design of materials for biosensors and environmental applications"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of \"Study of interaction energies between residues of the active site of Hsp90 and geldanamycin analogues using quantum mechanics/molecular mechanics methods\" describe the implementation of the methodology QSAR to explain the biological activity of geldanamycin analogues. Overall, the design of the calculus is good, and the results are of reasonable quality. There are several points that the authors should address before indexing in F1000Research:\nThe authors must clearly explain the contribution to the state of the art of this study. Apparently, the QSAR methodology doesn’t have new information, if there is something new in it, this should be highlighted in the summary and conclusion. It is possible that the novelty of the study is the explanation of how the geldanamycin analogues interact with Hsp90. Please emphasize this.\n\nThe authors generate models with 43 of 48 analogues that were reported in reference 21. The other 5 are not used because they do not report their IC50 value. Why is the activity of these 5 not predicted?\n\nFigures 7, 9 11-16: the y-axes have no units (EI?).\n\nApparently, the authors do not indicate the method of DFT used for the calculations of reactivity descriptors.\n\nThe direct application of this study is the use of models to predict or propose new molecules with more activity. As a recommendation, the authors should propose other molecules of this type that present greater activity. It would be more interesting to study for those who are working on the synthesis of these molecules.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5374",
"date": "16 Apr 2020",
"name": "Ricardo Vivas-Reyes",
"role": "Author Response",
"response": "1. The authors must clearly explain the contribution to the state of the art of this study. Apparently, the QSAR methodology doesn’t have new information, if there is something new in it, this should be highlighted in the summary and conclusion. It is possible that the novelty of the study is the explanation of how the geldanamycin analogues interact with Hsp90. Please emphasize this.Answer: Thanks for your comments. The information was included in the manuscript. 2. The authors generate models with 43 of 48 analogues that were reported in reference 21. The other 5 are not used because they do not report their IC50 value. Why is the activity of these 5 not predicted?- 5. The direct application of this study is the use of models to predict or propose new molecules with more activity. As a recommendation, the authors should propose other molecules of this type that present greater activity. It would be more interesting to study for those who are working on the synthesis of these molecules.Answer: Thanks for your comment. The information was not included because the objective of the study was to know that amino acid residues were key to the biological activity of the compounds and that groups of substituents on C-11 and C-17 improved the activity. The prediction of the activities is intended for a second article, where new analogs are also proposed.3. Figures 7, 9 11-16: the y-axes have no units (EI?).Answer: Thanks for your comments. The information was included in the manuscript. 4. Apparently, the authors do not indicate the method of DFT used for the calculations of reactivity descriptors.Answer: Was done."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2040
|
https://f1000research.com/articles/9-262/v1
|
16 Apr 20
|
{
"type": "Method Article",
"title": "PRESa2i: incremental decision trees for prediction of Adenosine to Inosine RNA editing sites",
"authors": [
"Alif Choyon",
"Ashiqur Rahman",
"Md. Hasanuzzaman",
"Dewan Md Farid",
"Swakkhar Shatabda",
"Alif Choyon",
"Ashiqur Rahman",
"Md. Hasanuzzaman",
"Dewan Md Farid"
],
"abstract": "RNA editing is a very crucial cellular process affecting protein encoding and is sometimes correlated with the cause of fatal diseases, such as cancer. Thus knowledge about RNA editing sites in a RNA sequence is very important. Adenosine to Inosine (A-to-I) is the most common of the RNA editing events. In this paper,we present PRESa2i, a computation prediction tool for identification of A-to-I RNA editing sites in given RNA sequences. PRESa2i uses a simple, yet effective set of sequence based features generated from RNA sequences and a novel feature selection technique. It uses an incremental decision tree algorithm as the classification algorithm. On a standard benchmark dataset and independent set, it achieves 86.48% accuracy and 90.67% sensitivity and significantly outperforms state-of-the-art methods. We have also implemented a web application based on PRESa2i and made it available freely at: http://brl.uiu.ac.bd/presa2i/index.php. The materials for this paper are also available to use from: https://github.com/swakkhar/RNA-Editing/.",
"keywords": [
"RNA Editing Sites",
"Sequence Based Features",
"Feature Selection",
"Incremental Decision Tree"
],
"content": "Introduction\n\nRNA editing is a process by which insertion, deletion or substitution of base nucleotides happen within the RNA. Such editing changes occur within the cell meaning nucleotide sequences are altered. This may cause alteration in the peptide sequence, which could be entirely different from that encoded in the DNA. Adenosine to Inosine (A-to-I) is the most common of all RNA editing events. Apart from making genetic alterations and resulting in codon changes A-to-I is often responsible for several intracellular operations like alternative splicing1. A-to-I is found to have its effect on RNA secondary structural changes2. Moreover, it is found to be in correlation with cancer formation3. Thus, research and identification of RNA editing is very important.\n\nExperimental methods like RNA-Seq4 are effective means for identification of A-to-I sites. However, they are time consuming. Moreover, as known RNA-editing sites are available, computational methods for prediction of RNA editing sites are becoming more relevant in this era of knowledge and data discovery. Various computational methods are proposed in the literature for prediction of RNA editing sites, including Genetic Algorithms (GA)5, Support Vector Machines (SVM)6, Logistic Regression (LR)7 and Deep Learning8. PREPACT was proposed as a computational tool for C-to-U and U-to-C RNA editing site prediction for plant species9. Another method was presented by 10 for prediction of C-to-U editing sites using biochemical and evolutionary information.\n\nSeveral machine learning based prediction methods are proposed in the literature of A-to-I RNA editing site identification in recent years. PAI was proposed by 11 that used pseudo nucleotide compositions as features with a SVM classification algorithm. They proposed and built two benchmark datasets derived from the Drosophila genome based on the work by 12. In a subsequent work, they proposed iRNA-AI6 using general form of pseudo nucleotide composition (PseKNC) with SVM and further improved the previous results on A-to-I datasets. A prediction method iRNA-3typeA13 was proposed to detect and identify three types of RNA-modifications at Adenosine sites. In a recent work, auto-encoders were used to develop PAI-SAE8 for prediction of A-to-I sites.\n\nIn this paper, we propose and present PRESa2i, a novel prediction method for A-to-I RNA Editing sites. In our proposed method, we have used the Hoeffding tree, an incremental decision tree for classification of samples. We have used sequence based features extracted from RNA samples collected as positive and negative instances in the dataset. We have used a novel feature selection method to select only the top 179 features. On a standard benchmark dataset PRESa2i has achieved a significant accuracy of 86.48% and on the independent test set it has a significant sensitivity of 90.67%. We have also made our method freely available for use by researchers at: http://brl.uiu.ac.bd/presa2i/index.php.\n\n\nMethods\n\nPRESa2i is trained with a dataset that has sequence based features. A novel feature selection algorithm is applied to select the top features and incremental decision tree algorithms are learned. The model is built on a standard benchmark training set and the performance of the model is tested using an independent set. As suggested by 14 and followed by many researchers15–18, we follow five steps for establishing any good tool for attribute prediction of biological entities: i) selection of benchmark datasets, ii) feature generation, iii) selection of appropriate algorithm, iv) evaluation methods, and v) establishment of a web server.\n\nThe first step in any machine learning based supervised prediction task is to select a standard set of data. The RNA A-to-I editing site prediction problem is formulated as a binary classification problem, where Adenine in a RNA sequence or subsequence is labeled with two classes: A-to-I or positive samples and non A-to-I or negative samples. A dataset can be formally defined as the following:\n\n\n\nHere, S denotes the dataset and positive and negative datasets are denoted by S+ and S– respectively. The datasets that we have used is first used in 11 based on the work in 12. The original dataset constructed in 12 contained 127 A-to-I editing sites with sequences and 127 non A-to-I editing sites with sequences. These sequences were obtained by sequencing wild-type and ADAR-deficient D. melanogaster DNA and RNA. After removing the redundant sequences, a benchmark dataset was constructed that contained 125 positive or A-to-I sites with sequences and 119 negative or non A-to-I sequence sites. Each of these RNA sequences in the dataset are 51 nucleotides long. From herein, we refer to this dataset as benchmark dataset.\n\nAn independent test set was constructed by further analyzing the sequences of D. melanogaster by 19. This set contained 51 nucleotide long 300 positive A-to-I sites with sequence. A summary of the datasets are presented in Table 1. Note that the benchmark dataset is balanced, while the independent dataset contains only positive sequences. Both datasets used are available here: https://github.com/swakkhar/RNA-Editing/.\n\nAny sample in the dataset is a sequence of RNAs. These are all 51 nucleotide long RNA strings. All of these strings are formed by taking symbols from the RNA alphabet, Σ = {A, C, G, U}. Formally a RNA sequence string R ∈ S can be formulated as below:\n\n\n\nHere, Ni is a nucleotide symbol and L = 51 is the length of the RNA sequence. We have extracted three groups of features from each sequence. Thus each sample in the dataset corresponds to a feature vector containing three groups of features, as below:\n\nThe three groups of features that were considered in the PRESa2i method are: k-mer compositions, gapped k-mer compositions and other statistical features. The first two types of features are widely used in the literature for solving other problems as well.\n\n1. k-mer Compositions: k-mers are sequences of length k drawn from the alphabet Σ. Compositions are calculated as the frequency of the different length k-mers normalized by sequence length. This is a widely used feature in the literature15,20. We have used k-mers with value of k = 1, 2, 3, 4. Thus the total number of features in this group was 4+16+64+256=340.\n\n2. Gapped k-mers: We have used gapped k-mer compositions as features. These features were previously used in the literature15,21 and are extended from the ideas of gapped k-mer composition22 or gapped di-peptide composition23. They are normalized frequency of k-mers with gaps in between them. We have considered gaps, g = 1, 2, 3, · · · , 10 in this paper and considered up to k-mers with k = 2, 3, 4. Here, the total number of features is 160+640+640+2560=4000.\n\n3. Other statistical features: We have used other statistically derived features, ratio of start and end codons and distribution of bases, as features. In total, there were five features from this group. The start codon is AUG and end codons are UGA, UAA and UAG. We have used the ratio of these codons. Also there are four nucleotides or bases: A, U, G, C. We have converted the presence of these bases into a distribution and used that as a feature.\n\nTotal number of features generated using the feature extraction technique was 4345. The feature generation technique is simple since they are extracted directly from RNA sequences. After the features are selected, we have used a hybrid feature selection technique. Our feature hybrid feature selection method is a multi-step step method where the first step is a wrapper method followed by consecutive filter methods. In the first phase of feature selection we have used best first search (BFS). It is an incremental feature selection procedure that adds or deletes one feature at a time and finds the optimal set of features. After the first phase the selected features were then sent for a single pass classification done by a logistic function (LF). For each class labels, the features with positive weights were considered and others were filtered out in this phase. In the last step, we used ensemble of random decision trees and added most significant features found by the randomly generated features which were left in the previous step to get the final set of features. Figure 1 shows the steps of the feature selection technique used by the PRESa2i method.\n\nWe have used Hoeffding tree24, an incremental decision tree algorithm as the classification engine for PRESa2i. Though its more applicable to data streams, the performance of this classifier is nearly the same as non-incremental learning algorithms. This algorithm starts with a single leaf decision tree and in each iteration for all examples it uses the Hoeffding bound to split the node, recursively selecting one attribute each time based on evaluative functions. The leafs are decided using adaptive naive Bayes technique.\n\nWe have used four metrics for the binary classification problem. These are Accuracy, Sensitivity (Sn), Specificity (Sp) and Mathew’s Correlation Coefficient (MCC). These metrics were selected to conform with previous methods8,11. For a binary classification method, let T P denote the number of true positives, T N denote the number of true negatives, F P denote the number of false positives and F N denote the number of false negatives. Now the four metrics are defined as follows:\n\n\n\n\n\n\n\n\n\nNote that, the first three metrics have the values in the range [0,1], where a value equal or near 0 means a bad classifier and a value near 1 means a good classifier. However, MCC have values in range [-1,1]. We have also used Receiver Operating Characteristic (ROC) curve to show the performances of different classifiers. It plots true positive rate against false positive rate at different thresholds of a probabilistic classification method.\n\n\nResults and discussion\n\nIn this section, we present the details of experimental results for our proposed method, PRESa2i. All the algorithms and programs were developed using Java 8 standard edition and Weka library25. All the experiments were done 10 times and only the average results are reported.\n\nIn this section, we provide the experimental results for comparisons with other state-of-the-art methods with our method PRESa2i. We have considered two previous state-of-the-art methods for comparison with our method: PAI proposed by 11 and PAI-SAE proposed by 8. We have used accuracy, sensitivity, specificity and MCC to compare the results. We have not run PAI and PAI-SE, rather as all these methods are tested on the same benchmark dataset, we have taken the results as being reported in the respective publications. Table 2 presents the results on the benchmark dataset. In this table, best values in each criteria are shown in bold fonts.\n\nFrom the values reported in Table 2, we can see that PRESa2i achieves higher values in all the performance metrics compared with PAI and PAI-SAE. In case of accuracy our method is 6.89% and 4.51% more accurate than PAI and PAI-SAE, respectively. It also outperforms both of the methods in terms of MCC and sensitivity. In case of specificity its performance is significantly improved compared to other two methods. To further assess the performance of our method we used an independent test set. Note that, while constructing the independent test set CD-HIT was used to remove sequences with higher similarity. We used the same cutoff as being used in PAI proposed by 11. In the case of the independent test set as there were no negative samples, and the only metric applicable was sensitivity. We report the sensitivity of PAI and PRESa2i in Table 3.\n\nNote that, the sensitivity of our method on the benchmark dataset was not a result of overfit and hence 272 samples out of 300 samples were correctly predicted in the independent test set by PRESa2i compared to 247 correctly predicted by PAI. Thus, we can conclude that our method achieved superior performances in both benchmark dataset and independent test set over these current state-of-the-art methods.\n\nWe have proposed a multi-stage novel feature selection method. To show the effectiveness of our feature selection method, we have used accuracy, sensitivity and specificity as performance measures. We have compared the performance of the feature selection method with that of ‘Using All Features’ and with that of ‘Reduced Using BFS+LF’. Results are reported in Table 4. We have used six classifiers: Naive Bayes Classifier, AdaBoost Classifier, Hoeffding Tree, Random Forest Classifier, Support Vector Machines and Bagging.\n\nThe bold fonts show the best values of accuracy, sensitivity and specificity for each of the classifiers on the different set of features selected at different stages of feature selection. From this, we can note the effectiveness of the over-all feature selection procedure. Here, we can see that, using best first search and logistic function based feature selection in the first phase generates features that improves the performance of all the classifiers compared to no feature selection. On the other hand, when we further refine the features using decision tree based significance test, all the measures improve for four of the classifiers which is an indication of the effectiveness of the later stage of feature selection.\n\nWe can note that among the six classifiers used in our experiments as shown in Table 4, AdaBoost classifier and Hoeffding Tree are the two best classifiers, followed by SVM. In the case of SVM though the accuracy is higher compared to these two algorithms, the sensitivity is not as good as these two. Note that these are the results on the benchmark dataset. We have also shown the receiver operating characteristic curve for each of these classifiers in Figure 2.\n\nTo make the selection of the final classification algorithm, we also tested them on the independent dataset. In Table 5, the results on the independent test set in terms of sensitivity is presented. We see that, the performance of Hoeffding tree does not overfit the data in benchmark dataset and hence the accuracy is enhanced from 86.47% to 90.67% from benchmark dataset to the independent test set. Thus we chose Hoeffding tree as our final classifier and save the model learned by this algorithm for the prediction tool.\n\nWe have further investigated the effect of introducing negative instances in the independent test set. For this purpose, we have generated negative instances using a empirical distribution learned from the negative training dataset based on the distribution of the base nucleotides only. The generated negative instance are then added to the positive ones and the algorithms were again tested on this set. The results are reported in Table 6.\n\nFrom the results reported in Table 6, we note that addition of the negative data in the independent test set does not affect on the effectiveness of the features and the feature selection method and the model trained. Note that, the introduced negative instances are given in the repository at: https://github.com/swakkhar/RNA-Editing/.\n\nPlease note that the results presented in Table 6 shows the effect of introducing negative examples in the dataset. Note that these negative datasets are taken randomly and are not verified at all. We note that though the accuracy and sensitivity is still satisfactory, Hoeffding tree and AdaBoost fails in terms of specificity. However, it also indicates that there is scope to improve the performance by adding more negative samples in the training set and also to improve the independent test set by adding real negative examples instead of taking random samples.\n\nWe have also implemented a web server, based on the best classification model learned by the incremental decision tree and final selected set of features on the benchmark dataset. Our website was developed using PHP language on the server side using Java and Weka library to provide the prediction from backend. Our method is readily available for use from: http://brl.uiu.ac.bd/presa2i/ index.php.\n\nThe web server has two major components: an web interface and a background server application. A small brief on the implementation is given below:\n\n1. Web Interface: The web interface and its backend is implemented using PHP and boostrap. The PHP script takes the input of the RNA sequence and then calls the prediction server which is a Java program and again receives the output from the prediction server and shows them in the web area.\n\n2. Prediction Server: The prediction server is written in Java language. All the experiments are written in java language. The feature generation and selection part is implemented using Java. A program takes the input of the RNA sequence and generates the necessary features and then uses the Weka package for the classification of indentification engine. After the results are processed they are fed back to the Web interface where the results are shown. Interface between the Web Interface and the Java program is made using secured http connection.\n\nThe operation of the webserver is very simple. There are only one text field and two buttons. A screenshot of the developed web application is given in Figure 3. Here is a step by step procedure on thow to use the web application:\n\n1. First one has to put the input RNA sequences in the text area. If there is any mistake, pressing the ‘Clear’ button will clear the area.\n\n2. After the input is given in the text area, the predict button should be pressed. Thus a request would be made to the online server.\n\n3. The results for the query sequence will be shown just below the input pane. For the next entry, the process should be followed in the same manner starting from step 1.\n\n\nConclusion\n\nIn this article, we have proposed PRESa2i, a novel predictor for A-to-I RNA editing sites using sequence based features and incremental decision trees. Our method is based on a number of effective features selected using successive phases of best first search in incremental forward selection and ranking based on random decision trees and logistic regression. We have made all our datasets available online at: https://github.com/swakkhar/RNA-Editing/, and have also made our method available as an web application from: http://brl.uiu.ac.bd/presa2i/index.php. In the future, we would like to explore other RNA editing sites and develop layered or multi-class prediction system. We believe further enhancement of the dataset can improve the performance of the method to a great extent.\n\n\nData availability\n\nDatasets for this article available from: https://github.com/swakkhar/RNA-Editing/\n\nArchived datasets as at time of publication: https://zenodo.org/record/372722127\n\nLicense: CC0\n\n\nSoftware availability\n\nPRESa2i web application: http://brl.uiu.ac.bd/ presa2i/index.php\n\nSource code available from: https://github.com/swakkhar/RNA-Editing\n\nArchived source code as at time of publication: https://zenodo.org/record/372722127\n\nLicense: CC0",
"appendix": "References\n\nRueter SM, Dawson TR, Emeson RB: Regulation of alternative splicing by RNA editing. Nature. 1999; 399(6731): 75–80. PubMed Abstract | Publisher Full Text\n\nMaas Stefan: Gene regulation through RNA editing. Discov Med. 2010; 10(54): 379–386. PubMed Abstract\n\nPaz N, Levanon EY, Amariglio N, et al.: Altered adenosine-to-inosine RNA editing in human cancer. Genome Res. 2007; 17(11): 1586–1595. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009; 10(1): 57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThompson J, Gopal S: Genetic algorithm learning as a robust approach to RNA editing site prediction. BMC Bioinformatics. 2006; 7(1): 145. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen W, Feng P, Yang H, et al.: iRNA-AI: identifying the adenosine to inosine editing sites in RNA sequences. Oncotarget. 2017; 8(3): 4208–4217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiong H, Liu D, Li Q, et al.: RED-ML: a novel, effective RNA editing detection method based on machine learning. GigaScience. 2017; 6(5): 1–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiao X, Wang P, Xu Z, et al.: Pai-sae: Predicting adenosine to inosine editing sites based on hybrid features by using spare autoencoder. In IOP Conference Series: Earth and Environmental Science. 2018; 170(5): 052018. IOP Publishing. Publisher Full Text\n\nLenz H, Knoop V: PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation. Bioinform Biol Insights. 2013; 7: 1–19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDu P, Li Y: Prediction of C-to-U RNA editing sites in plant mitochondria using both biochemical and evolutionary information. J Theor Biol. 2008; 253(3): 579–586. PubMed Abstract | Publisher Full Text\n\nChen W, Feng P, Ding H, et al.: PAI: Predicting adenosine to inosine editing sites by using pseudo nucleotide compositions. Sci Rep. 2016; 6: 35123. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSt Laurent G, Tackett MR, Nechkin S, et al.: Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila. Nat Struct Mol Biol. 2013; 20(11): 1333–9. PubMed Abstract | Publisher Full Text\n\nChen W, Feng P, Yang H, et al.: iRNA-3typeA: Identifying Three Types of Modification at RNA's Adenosine Sites. Mol Ther Nucleic Acids. 2018; 11: 468–474. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChou KC: Some remarks on protein attribute prediction and pseudo amino acid composition. J Theor Biol. 2011; 273(1): 236–247. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRahman S, Aktar U, Jani R, et al.: iPromoter-FSEn: Identification of bacterial σ70 promoter sequences using feature subspace based ensemble classifier. Genomics. 2019; 111(5): 1160–1166. PubMed Abstract | Publisher Full Text\n\nRayhan F, Ahmed S, Shatabda S, et al.: iDTI-ESBoost: Identification of Drug Target Interaction Using Evolutionary and Structural Features with Boosting. Sci Rep. 2017; 7(1): 17731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChowdhury SY, Shatabda S, Dehzangi A: iDNAProt-ES: Identification of DNA-binding Proteins Using Evolutionary and Structural Features. Sci Rep. 2017; 7(1): 14938. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUddin MR, Sharma A, Farid DM, et al.: EvoStruct-Sub: An accurate Gram-positive protein subcellular localization predictor using evolutionary and structural features. J Theor Biol. 2018; 443: 138–146. PubMed Abstract | Publisher Full Text\n\nYu Y, Zhou H, Kong Y, et al.: The Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection. PLoS Genet. 2016; 12(7): e1006191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShatabda S, Saha S, Sharma A, et al.: iPHLoc-ES: Identification of bacteriophage protein locations using evolutionary and structural features. J Theor Biol. 2017; 435: 229–237. PubMed Abstract | Publisher Full Text\n\nAl Maruf MA, Shatabda S: iRSpot-SF: Prediction of recombination hotspots by incorporating sequence based features into Chou's Pseudo. Genomics. 2019; 111(4): 966–972. PubMed Abstract | Publisher Full Text\n\nGhandi M, Mohammad-Noori M, Beer MA: Robust k-mer frequency estimation using gapped k-mers. J Math Biol. 2014; 69(2): 469–500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang J, Su EC, LO A, et al.: PSLDoc: Protein subcellular localization prediction based on gapped-dipeptides and probabilistic latent semantic analysis. Proteins. 2008; 72(2): 693–710. PubMed Abstract | Publisher Full Text\n\nHulten G, Spencer L, Domingos P: Mining time-changing data streams. In ACM SIGKDD Intl. Conf. on Knowledge Discovery and Data Mining. ACM Press. 2001; 97–106. Publisher Full Text\n\nHall M, Frank E, Holmes G, et al.: The weka data mining software: an update. ACM SIGKDD explorations newsletter. 2009; 11(1): 10–18. Publisher Full Text\n\nChen XX, Tang H, Li WC, et al.: Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition. Biomed Res Int. 2016; 2016: 1654623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nswakkhar: swakkhar/RNAEditing:Pres-a2i (Version v1.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3727221"
}
|
[
{
"id": "62452",
"date": "19 May 2020",
"name": "Avdesh Mishra",
"expertise": [
"Reviewer Expertise Artificial Intelligence and Machine Learning",
"Data Science",
"Bioinformatics",
"Computational and Structural Biology",
"Biomedical Informatics",
"Evolutionary Computation",
"Information Retrieval and Text Mining",
"and Big Data."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, authors present PRESa2i, a computation prediction tool for identification of A-to-I RNA editing sites in given RNA sequences. PRESa2i uses a simple, yet effective set of sequence based features generated from RNA sequences and a novel feature selection technique. It uses an incremental decision tree algorithm as the classification algorithm.\nBelow are my comments and suggestions:\nAuthors should specify which method the proposed method outperforms \"significantly outperforms state-of-the-art methods\" is a very general statement. In near future there will be another method that would outperform this method.\n\nI did not find any example RNA sequence when I clicked on \"click here\" link on the authors webserver.\n\nThe webserver is not working. I tried to run the webserver with an example RNA Sequence. But I got \"Login Failed\" message.\n\nDoes authors method achieves 86.48% accuracy and 90.67% sensitivity on benchmark or independent test data set? This is not clear from the sentence below from the abstract: \"On a standard benchmark data set and independent set, it achieves 86.48% accuracy and 90.67% sensitivity and significantly outperforms state-of-the-art methods.\" Use 'respectively' would help.\n\nIn sentence \"Another method was presented by 10 for prediction of C-to-U editing sites using biochemical and evolutionary information.\" does '10' refer to a reference? I found similar issues at other locations in the manuscript. Please review the entire manuscript for reference style.\n\nCorrect sentence grammar \"The datasets that we have used is first used in 11 based on the work in 12.\". Replace 'is' with 'was'.\n\nIs there a specific reason why independent test dataset does not contain negative samples?\n\nAuthors should to clearly specify what kind of features were extracted from the distribution of A, C, G, and U. Also how did they obtain the distribution of the A, C, G, and U.\n\nPlease check the grammar and remove the redundant words from this sentence: \"Our feature hybrid feature selection method is a multi- step step method where the first step is a wrapper method followed by consecutive filter methods.\"\n\nIt will be better if authors put some more light on feature selection approach. For example, how add or delete of one feature at a time works? Also, this sentence \"In the last step, we used ensemble of random decision trees and added most significant features found by the randomly generated features which were left in the previous step to get the final set of features.\" is difficult to read and understand. This will be helpful in replication of this work.\n\nHow many features were finally selected after the Hybrid feature selection? Authors need to include this information in the Hybrid Feature Selection section of the manuscript.\n\nWhat were the value of parameters for the incremental decision tree algorithm? Please specify the values of all the parameters that were used.\n\nAuthors are comparing their work with PAI and PAI-SAE which were published in year 2016 and year 2018. Doing a quick google search, I found a method EPAI-NC which was published in year 2019 that utilizes Locally Deep Support Vector Machines and same benchmark and independent test datasets. Compared to EPAI-NC the proposed PRESa2i shows poor performance. Authors need to compare their method with EPAI-NC as this is a method published in the recent past.\n\nAuthors should also compare their method with PAI-SAE and EPAI-NC on the independent test dataset as PAI-SAE and EPAI-NC are more recently published method compared to PAI.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No",
"responses": []
},
{
"id": "87702",
"date": "21 Jun 2021",
"name": "Shizuka Uchida",
"expertise": [
"Reviewer Expertise bioinformatics",
"biochemistry",
"developmental biology",
"molecular biology",
"RNA biology",
"non-coding RNAs",
"miRNAs",
"lncRNAs",
"circRNAs",
"A-to-I RNA editing",
"epitranscriptomics",
"RNA sequencing"
],
"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 present a new method to predict A-to-I RNA editing sites. This method is implemented in a web interface so that users can access freely. The method proposed is technically sound and valid, yet the rationale for developing such method is hampered by the fact that there are many bioinformatic tools available to detect (not predict) A-to-G changes from RNA sequencing (RNA-seq) data. Thus, the usage of the author’s web interface is not of high priority to researchers; given that the cost for RNA-seq is not high anymore. More specific comments are listed below:\nMajor points\nThe authors must benchmark the performance of their tool to the known RNA editing sites deposited in databases, such as DARNED, RADAR, and REDIportal.\n\nThe authors must provide the results of other species, as most of the current research in A-to-I RNA editing is focused on human diseases.\n\nThis web tool is not functioning. When the known RNA edited sequence, TTCTCAACCTCCTGGGCTCTTGTGATCCTCCTGTCTTA (from PMID: 27595325), was pasted onto the field, Enter the query RNA sequences here, and the [Predict] button is pressed, the following message appears: “Login Failed”. Thus, this reviewer cannot evaluate the actual performance of this tool that the authors developed.\nMinor points\n(1) From the URL (http://brl.uiu.ac.bd/presa2i/index.php#), nothing happens when “ • Don't have any RNA string click here.” is pressed.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "91074",
"date": "17 Aug 2021",
"name": "Xiaoyong Pan",
"expertise": [
"Reviewer Expertise Bioinformatics",
"RNA-protein interaction prediction"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a computational method for predicting A-to-I RNA editing sites using sequence-based features and incremental decision trees, and they show that the proposed method yields better performance. However, there are limited novelties in this manuscript.\nHow is sequence similarity between the training set and test set? The redundant sequences in the test set should be removed.\n\nDeep learning has been widely used in genomic sequences. The authors should compare the proposed method with the CNN-based method which uses a one-hot encoded sequence as input.\n\nIn Table 3, for the independent test set, why is only sensitivity reported? Are other metrics of the proposed method worse than the baseline method? It is bad to only keep positive samples in the independent test set.\n\nIn Figure 2, the AUC value should be added.\n\nHow is the performance of XGBoost on this task? This classifier has been shown to perform well on many tasks.\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-262
|
https://f1000research.com/articles/9-209/v1
|
25 Mar 20
|
{
"type": "Case Report",
"title": "Case Report: Ectopic third molar in the maxillary sinus with infected dentigerous cyst assessed by cone beam CT",
"authors": [
"Khairy Elmorsy",
"Lubna K. Elsayed",
"Sara M. El Khateeb",
"Khairy Elmorsy",
"Lubna K. Elsayed"
],
"abstract": "Ectopic development of teeth in nondental areas is uncommon, especially in the maxillary sinus. A panoramic radiograph is the routine diagnostic radiographic examination performed for this type of eruption, although cone beam computed tomography (CBCT) is highly recommended for further localization of the ectopic tooth and assessment of the characteristics of any associated lesion before a surgical procedure. We report a case of a 13-year-old female student who presented with purulent discharge posterior to the upper right second molar with a bad taste and foul odour. Radiographic examination revealed a maxillary third molar tooth located at the posterosuperior aspect of the right maxillary sinus with a hyperdense lesion surrounding the crown, obliterating the sinus cavity. Both the tooth and dentigerous cyst were surgically removed under general anaesthesia through Caldwell-Luc antrostomy. After a three-month follow-up, the patient was symptom free and had an uneventful recovery. The rare and critical location of the reported third molar along with the infected dentigerous cyst indicates its complete enucleation to avoid complications as recurrence or malignant transformation.",
"keywords": [
"Ectopic",
"tooth Eruption",
"Maxillary Sinus",
"Molar",
"Cone Beam CT"
],
"content": "Introduction\n\nAn ectopic eruption is a condition characterized by the presence of a tooth in a non-dentate area, distant from its usual anatomical location. The aetiology of an ectopic eruption can be unclear; however, several theories have been put forward to describe the rise of this condition, such as: developmental disturbances like cleft palate; pathological processes like large cysts, which displace tooth buds to other areas; odontogenic and rhinogenic infections; or iatrogenic activity1–4.\n\nThe maxillary sinus is an unusual location for an ectopic eruption, although some cases have been reported with unconventional management approaches5–7.\n\nEctopic eruption reporting is rare; this case reports the unusual location of the eruption as well as the radiographic assessment and the surgical approach and is a contribution to the literature on this topic.\n\n\nCase report\n\nA 13-year-old Caucasian female student reported to the oral and maxillofacial surgery clinic with the chief complaint of purulent discharge oozing just distal to the upper right second molar, with a bad taste and foul odour, that started two weeks before seeking treatment. Upon clinical examination, the patient had no intraoral or extraoral swelling, and there was a full complement of teeth on that arch except for teeth #18 and #28. All teeth were firm, vital and non-carious. A panoramic radiograph revealed ectopic eruption of the right maxillary third molar in the maxillary sinus with hyperdense lesion surrounding its crown and obliterating the sinus cavity (Figure 1).\n\nPreoperative panoramic radiograph showing an ectopic eruption of the right maxillary third molar in a superior position near pterygomaxillary fissure inside the right maxillary sinus and surrounded by well-defined hyperdense lesion obliterating the right maxillary sinus cavity.\n\nFurther radiographic investigation using cone beam computed tomography (CBCT) determined the exact location of the maxillary molar and the lesion extension since the molar was seen in close proximity to the infraorbital rim. CBCT showed the ectopic third molar with incompletely formed roots located in the posterosuperior aspect of the right maxillary sinus with a close approximation to the orbital floor superiorly and pterygoid plates posteriorly (Figure 2a). The third molar was surrounded by a well-defined corticated hyperdense lesion measuring 23×36×35mm, occupying almost the whole cavity of the right maxillary sinus and causing mediolateral expansion of the alveolar ridge. The CBCT also revealed the destructive effect of the associated pericoronal lesion on the right maxillary sinus floor and buccal cortical plate, distal to tooth #17, causing oroantral communication, which explains the reason for the purulent discharge, the chief complaint of the patient (Figure 2b, 2c, 2d and 2e). Differential diagnosis of the detected lesion was dentigerous cyst, odontogenic keratocyst or Gorlin cyst. Based on the clinical and radiographical examination, the surgical removal of the ectopic third molar along with cyst enucleation was planned through an intraoral approach under general anaesthesia induced via nasopharyngeal intubation. Induction of anaesthesia was achieved with propofol 2mg/kg, fentanyl 1µgm and atracurium 0.5 mg/kg, then maintained with propofol infusion 200µgm/kg/min, fentanyl 1µgm/kg/hour and atracurium 0.1 mg/kg every 20 minutes.\n\nMultiplanar cone beam computed tomography (CBCT) sections: (a) three-dimensional; (b) sagittal; (c, d) axial; (e) coronal slices showing the posterosuperior position of the ectopic right third maxillary molar inside the maxillary sinus with associated pericoronal hyperdense lesion and radiographic evidence of oroantral communication due to its destructive effect on the maxillary sinus floor and the alveolar ridge.\n\nAn oral and maxillofacial surgeon with 15 years of experience performed lateral sinus antrostomy, utilizing a standard Caldwell-Luc approach, with a bony window created in the anterolateral wall of the maxillary sinus (Figure 3a). An incision was made through the Schneiderian membrane to enter the maxillary sinus. The cystic lining was identified, and the pus was drained prior to the complete removal of the cystic lining and extraction of the ectopic maxillary third molar (Figure 3b). The antrum was thoroughly irrigated, and the cystic lining was placed in a 10% buffered formalin solution for subsequent histopathological examination and final diagnosis. The flap was closed with chromic catgut sutures with good approximation of edges. Postoperative antibiotics were given to the patient in form of 500 mg of amoxicillin every eight hours for a minimum of five days with an analgesic (325mg acetaminophen every four hours per day), also one dose of betamethasone 4 mg was given to prevent postoperative edema of the cheek, and the patient was instructed not to blow her nose for two weeks.\n\n(a) Perioperative image showing the bony window created in the anterior wall of the sinus. (b) Underdeveloped ectopic molar with cystic lining.\n\nHistopathologic examination reported a cystic cavity lined by a thin non-keratinized stratified squamous epithelium. Part of the epithelial lining showed hyperplasia due to inflammation and long-standing lesion, and a connective tissue wall infiltrated with chronic inflammatory cells and composed of fibroblasts, collagen fibres and blood vessels, suggesting an infected dentigerous cyst (Figure 4).\n\n(a, b) Photomicrograph of infected dentigerous cyst (×4, ×10). (c) Photomicrograph of infected dentigerous cyst showing thin non-keratinized epithelium (×4).\n\nA panoramic radiograph and CBCT were performed three months after the surgery. CBCT axial cuts revealed some bone formation in the mediolateral dimension when compared to the preoperative radiographs, which indicates that the bone is in the healing process (Figure 5a).\n\nMultiplanar cone beam computed tomography (CBCT) slices: (a) axial cut showing bone formation medially; (b, c) sagittal and coronal cuts showing discontinuity of the antral floor and part of the anterior antral wall with oroantral communication starting distal to tooth #16.\n\nHealing appeared to be better clinically than radiographically, the bone requiring longer to time form and be detected radiographically. There is improved bone healing in younger patients, and so we expect a quick healing process with our patient.\n\nPostoperative CBCT scans showed opacification and mucosal lining thickening of the right maxillary sinus, as well as continued discontinuity of the posterolateral floor and part of the anterior wall of the right maxillary sinus (Figure 5b and 5c).\n\n\nDiscussion\n\nEctopic eruption of teeth into dentate regions is relatively common, but such a condition in non-dentate areas like the mandibular condyle, sigmoid notch, or nasal cavity is rare, and the exact aetiology of this phenomenon is not clear8.\n\nOccasionally, the tooth may erupt into the maxillary sinus and could be asymptomatic9 or cause symptoms such as sinusitis or swelling, facial pain, rhinorrhoea, nasal obstruction and headache10. In the present case, the patient presented with purulent discharge distal to upper second molar and panoramic radiograph showed an ectopic third molar associated with a pericoronal lesion.\n\nSome studies report that panoramic radiography is the radiographic examination of choice in ectopic cases because of its accuracy in detecting these structures and the low level of radiation to which the patient is subjected6–11. However, the major disadvantage of two-dimensional panoramic radiography is the difficulty in interpreting the exact location of the ectopic tooth and the associated pathology because of the superimposition of different bony structures12.\n\nCBCT presents an accurate three-dimensional imaging modality that offers highly diagnostic images with a sub-millimetre resolution, short scanning time and reduced radiation dose up to 15 times less than multi-slice CT scans13.\n\nThe CBCT assessment of our case allowed us to precisely locate the ectopic molar and showed the exact extension and effect of the associated pericoronal lesion on the surrounding structures. This allowed the preoperative identification of the cause of the purulent discharge and aided the surgeon during the surgical procedure, mainly to avoid any surgical complications due to the proximity to the orbital floor and pterygoid plates.\n\nAlqerban et al. and Botticelli et al. reported better accuracy and significant interobserver agreement in the evaluation of ectopic canine location when using CBCT compared to panoramic radiography11,14. The smallest field of view matched the clinical indication, recommended to minimize the radiation exposure to the patient15.\n\nIn our case, histopathologic examination showed an associated infected dentigerous cyst with the ectopic molar, similar to what has been described previously in the literature, where the dentigerous cysts are the most common pathologic lesion associated with ectopic eruptions16.\n\nCaldwell-Luc antrostomy and full enucleation of the cyst was performed on our patient, as it provides a direct view of the maxillary sinus and allows instrumentation, irrigation and removal of large objects, making it the treatment of choice for surgery at the maxillary sinus17.\n\nVarious techniques for managing ectopic teeth are mentioned in the literature, such as endoscopic assisted procedures18,19, as well as extra and trans-oral approaches20. Endoscopic approaches have less morbidity post and intraoperatively21. Liau et al. utilized the endoscopically assisted Caldwell-Luc approach for safer instrumentation of the supero-lateral aspect of the maxillary sinus, allowing direct line access for visualization of potential orbital floor defects. He concluded that this approach allowed accurate visualization of inaccessible areas of the maxillary sinus with the ability to perform surgical intervention under direct line access as required19.\n\nIn the present paper, we report a case of infected dentigerous cyst associated with an ectopic molar within the maxillary sinus. Surgical removal for both was performed, and the entire pathologic antral tissue was removed completely and assessed histologically. There were no post-surgical complications, and the patient's postoperative healing was satisfactory.\n\n\nConclusion\n\nEctopic tooth eruptions in the maxillary sinus are rare. However, its association with dentigerous cysts is relatively common. A standard Caldwell-Luc approach is the management of choice to surgically extract the ectopic tooth along with excision of the pathologic tissue to avoid sinus or ophthalmic complications. Asymptomatic cases should be managed with a similar protocol due to their tendency to form cysts or malignancies.\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 parent of the patient.",
"appendix": "References\n\nBodner L, Tovi F, Bar-Ziv J: Teeth in the maxillary sinus--imaging and management. J Laryngol Otol. 1997; 111(9): 820–824. PubMed Abstract | Publisher Full Text\n\nO’meara WF: Ectopic Eruption Pattern in Selected Permanent Teeth. J Dent Res. 1962; 41(3): 607–616. Publisher Full Text\n\nLee FP: Endoscopic extraction of an intranasal tooth: A review of 13 cases. Laryngoscope. 2001; 111(6): 1027–1031. PubMed Abstract | Publisher Full Text\n\nBaykul T, Doğru H, Yasan H, et al.: Clinical impact of ectopic teeth in the maxillary sinus. Auris Nasus Larynx. 2006; 33(3): 277–281. PubMed Abstract | Publisher Full Text\n\nGoh YH: Ectopic eruption of maxillary molar tooth--an unusual cause of recurrent sinusitis. Singapore Med J. 2001; 42(2): 080–081. PubMed Abstract\n\nChagas Júnior OL, Moura LB, Sonego CL, et al.: Unusual Case of Sinusitis Related to Ectopic Teeth in the Maxillary Sinus Roof/Orbital Floor: A Report. Craniomaxillofacial Trauma Reconstr. 2016; 09(03): 260–263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBello SA, Oketade IO, Osunde OD: Ectopic 3rd Molar Tooth in the Maxillary Antrum. Case Rep Dent. 2014; 2014: 620741. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFindik Y, Baykul T: Ectopic third molar in the mandibular sigmoid notch: Report of a case and literature review. J Clin Exp Dent. 2015; 7(1): e133–e137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKheir MK, Sheikhi M: Ectopic third molar in maxillary sinus: an asymptomatic accidental finding. Egypt J Otolaryngol. 2019; 35(2): 219–221. Reference Source\n\nLamb JF, Husein OF, Spiess AC: Ectopic molar in the maxillary sinus precipitating a mucocele: a case report and literature review. Ear Nose Throat J. 2009; 88(8): E6–E11. PubMed Abstract\n\nBotticelli S, Verna C, Cattaneo PM, et al.: Two- versus three-dimensional imaging in subjects with unerupted maxillary canines. Eur J Orthod. 2011; 33(4): 344–349. PubMed Abstract | Publisher Full Text\n\nLoubele M, Maes F, Schutyser F, et al.: Assessment of bone segmentation quality of cone-beam CT versus multislice spiral CT: a pilot study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006; 102(2): 225–234. PubMed Abstract | Publisher Full Text\n\nScarfe WC, Farman AG, Sukovic P: Clinical Applications of Cone-Beam Computed Tomography in Dental Practice. J Can Dent Assoc. 2006; 72(1): 75–80. PubMed Abstract\n\nAlqerban A, Jacobs R, Fieuws S, et al.: Comparison of two cone beam computed tomographic systems versus panoramic imaging for localization of impacted maxillary canines and detection of root resorption. Eur J Orthod. 2011; 33(1): 93–102. PubMed Abstract | Publisher Full Text\n\nSerrant PS, McIntyre GT, Thomson DJ: Localization of ectopic maxillary canines -- is CBCT more accurate than conventional horizontal or vertical parallax? J Orthod. 2014; 41(1): 13–18. PubMed Abstract | Publisher Full Text\n\nBuyukkurt MC, Omezli MM, Miloglu O: Dentigerous cyst associated with an ectopic tooth in the maxillary sinus: a report of 3 cases and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109(1): 67–71. PubMed Abstract | Publisher Full Text\n\nSivolella S, Ricci S, Busca M, et al.: Maxillary dentigerous cyst associated with an ectopic third molar in the maxillary sinus: a literature review and report of six consecutive cases. Oral Surg. 2014; 7(S1): 72–78. Publisher Full Text\n\nChristmas DA, Mirante JP, Yanagisawa E: Endoscopic view of a maxillary dentigerous cyst. Ear Nose Throat J. 2008; 87(6): 316. PubMed Abstract | Publisher Full Text\n\nLiau I, Lynch N, Hearn B, et al.: Endoscopically Assisted Modified Caldwell-Luc Approach to Enucleation of Dentigerous Cyst With Ectopic Tooth From the Maxillary Sinus. J Craniofac Surg. 2018; 29(6): e568–e570. PubMed Abstract | Publisher Full Text\n\nRai A, Rai NJ, Rai MA, et al.: Transoral removal of ectopic maxillary third molar situated superiorly to maxillary antrum and posteroinferiorly to the floor of orbit. Indian J Dent Res. 2013; 24(6): 756–758. PubMed Abstract | Publisher Full Text\n\nGangadhara Somayaji K, Rajeshwary A, Ramlan S, et al.: Ectopic premolar tooth in the maxillary sinus: A case report and review of literature. Arch Med Health Sci. 2013; 1(1): 48–51. Publisher Full Text"
}
|
[
{
"id": "61698",
"date": "02 Apr 2020",
"name": "Najla Dar-odeh",
"expertise": [
"Reviewer Expertise Oral Medicine."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study describes a case of a purulent infection that originated in a dentigerous cyst associated with an ectopic third molar in the maxillary sinus. Three diagnostic methods were utilized in this study. A panoramic radiograph was employed initially, followed by a more accurate imaging technique of CBCT to get more detailed information on the extent of the lesion, and to elicit a more accurate differential diagnosis. Finally a histopathological examination was done to achieve a final diagnosis. Successful treatment outcomes were accompanied by a satisfactory follow-up period, and this further increased the chances for a good prognosis.\nThe term “ectopic eruption” was used throughout the text, however, taking into consideration the age of the patient and the definition of “eruption”, it is preferable to replace it with the term “ectopic tooth”.\nLiterature review was thorough, however, several references were published more than a decade ago. It would be much improved by inclusion of more recent literature, especially case reports that tackle the topic of ectopic third molar in the maxillary sinus, and that take in consideration the young age of the patient.\nDiscussion: first paragraph mentions that exact aetiology is not clear, what do you think the aetiology for ectopic tooth is in your case?\n\nDiscussion: How many cases of ectopic teeth in the maxillary sinus were reported so far? And how many of them were for third molars?\nI thank authors for the informative manuscript and the solid treatment plan. I also encourage them to write a literature review on this 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": [
{
"c_id": "5395",
"date": "06 Apr 2020",
"name": "sara elkhateeb",
"role": "Author Response",
"response": "Comment No 1The term “ectopic eruption” was used throughout the text, however, taking into consideration the age of the patient and the definition of “eruption”, it is preferable to replace it with the term “ectopic tooth”.Reply to No 1:The term “eruption” means emission, ejection or discharge. Since the molar was clinically “visible” in the maxillary sinus, it is scientifically correct to describe it as “eruption” inside maxillary sinus.Also, the term “ectopic eruption” was previously reported in papers published in indexed journals in similar ectopic cases in maxillary sinus.Paper examples:-Sharma S, Chauhan JS. Bilateral ectopic third molars in maxillary sinus associated with dentigerous cyst.—A rare case report. International journal of surgery case reports. 2019 Jan 1; 61:298-301.-Lombroni LG, Farronato G, Santamaria G, Lombroni DM, Gatti P, Capelli M. Ectopic teeth in the maxillarysinus: A case report and literature review. Indian Journal of Dental Research. 2018 Sep 1;29(5):667.-Jalal H, Hicham S, Lahcen K, Karim EK. Dentigerous Cyst Associated with an Ectopic Third Molar in the Maxillary Sinus: Report of Cases and Review of Literature. Oral & Maxillofacial Pathology Journal. 2018 Jan 1;9(1). Comment No 2Literature review was thorough, however, several references were published more than a decade ago.Reply to No 2: We agree to some extent with this comment, reference no. 2 will be replaced with:Lombroni L, Farronato G, Santamaria G, Lombroni D, Gatti P, Capelli M. Ectopic teeth in the maxillary sinus: A case report and literature review. Indian J Dent Res. 2018;29(5):667.For the rest of references, we cannot make any changes in them as we think that having diversity in time ranges in the references and enriches our literature, as we included also 2013/14/18 and 19 articles.Comment No 3Discussion: first paragraph mentions that exact aetiology is not clear, what do you think the aetiology for ectopic tooth is in your case?Reply to No 3There is no exact etiology for ectopic teeth development, there are multiple theories to get closer to the exact cause. We will add the following statement to get closer to the etiology of developing ectopic molar in our case:\"In our case, we may suggest that the theory of the associated pathology with the third molar might have caused its ectopic location\".(To be added at the end of the first paragraph in discussion). Comment No 4Discussion: How many cases of ectopic teeth in the maxillary sinus were reported so far? And how many of them were for third molars?Reply to No 4According to latest review of literature 2018 (added ref no 2) there were 51 patients were observed, with higher prevalence of ectopic teeth in the third molars, 21 cases. Lombroni L, Farronato G, Santamaria G, Lombroni D, Gatti P, Capelli M. Ectopic teeth in the maxillary sinus: A case report and literature review. Indian J Dent Res. 2018;29(5):667."
}
]
},
{
"id": "61700",
"date": "03 Apr 2020",
"name": "Shadia Abdelhameed Elsayed",
"expertise": [
"Reviewer Expertise Oral and Maxillofacial 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\nThanks for this interesting, fully described, detailed, well written case.The manuscript needs minor revisions. Some points need to be declared.\nTitle Title need to be summarized and \"assessed by cone beam CT\" could be removed from the title. CT nowadays used routinely to diagnose the midface lesions for its difficult interpretation on plan panoramic films. Case reports are usually describing a very rare condition however dentigerous cyst in the maxillary sinus is not uncommon.\nFigure 3 is an intraoperative photo not perioperative image.\nUsually dentigerous cyst is diagnosed mainly by association with impacted tooth so the conclusion of the paper needs to be more accurate as this case is not ectopic eruption. The following paragraph is more accurately describing the present case: In the present paper, we report a case of infected dentigerous cyst associated with an ectopic molar within the maxillary sinus. Surgical removal for both was performed, and the entire pathologic antral tissue was removed completely and assessed histologically. There were no post-surgical complications, and the patient’s postoperative healing was satisfactory.\nIntroduction Ectopic eruption definition needs more justification as the present tooth is ectopic but associated with large maxillary cyst and pushed superiorly as result of cyst enlargement. Authors did not report any prevalence rate of the dentigerous cyst (which usually associated with impacted wisdom) in the maxillary sinus and also the rate for ectopic impacted teeth in the sinus.\nOperation The cystic lesion had discharge and was diagnosed as infected cyst, does the surgeon treat infection before opening the sinus especially as the cyst was communicate with the oral cavity and possibility of recurrent fistula is high? (Preoperative management of infected sinus is not mentioned).\nWhy is nasal anticongestant not prescribed as routine post sinus surgery (note that the sinus membrane appears still edematous in the CBCT)?\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": "5396",
"date": "06 Apr 2020",
"name": "sara elkhateeb",
"role": "Author Response",
"response": "Comment No 1 Title need to be summarized and \"assessed by cone beam CT\" could be removed from the title.Reply to No 1We prefer to keep the title as it is, we think its more enriching to the title to elaborate on the radiographic assessment of the case, especially that we noticed some ectopic cases were not evaluated using CBCT.Comment No 2 Case reports are usually describing a very rare condition however dentigerous cyst in the maxillary sinus is not uncommon.Reply to No 2Our report is focusing on the ectopic location of the molar “which is a rare condition” and being associated with dentigerous cyst.Comment No 3Figure 3 is an intraoperative photo not perioperative image.Reply to No 3The prefix “Peri” means “Intra or during”, so no change will be performed. FYI prefix “Pre” means “before”Comment No 4Usually dentigerous cyst is diagnosed mainly by association with impacted tooth so the conclusion of the paper needs to be more accurate as this case is not ectopic eruption.The following paragraph is more accurately describing the present case: In the present paper, we report a case of infected dentigerous cyst associated with an ectopic molar within the maxillary sinus. Surgical removal for both was performed, and the entire pathologic antral tissue was removed completely and assessed histologically. There were no post-surgical complications, and the patient’s postoperative healing was satisfactory.Reply to No 4a) The term “eruption” means emission, ejection or discharge. Since the molar was clinically “visible” in the maxillary sinus, it is scientifically correct to describe it as “eruption” inside maxillary sinus. Also, the term “ectopic eruption” was previously reported in papers published in indexed journals in same ectopic cases in maxillary sinus.Paper examples: Sharma S, Chauhan JS. Bilateral ectopic third molars in maxillary sinus associated with dentigerous cyst: A rare case report. International journal of surgery case reports. 2019 Jan 1; 61:298-301. Lombroni LG, Farronato G, Santamaria G, Lombroni DM, Gatti P, Capelli M. Ectopic teeth in the maxillary sinus: A case report and literature review. Indian Journal of Dental Research. 2018 Sep 1;29(5):667. Jalal H, Hicham S, Lahcen K, Karim EK. Dentigerous Cyst Associated with an Ectopic Third Molar in the Maxillary Sinus: Report of Cases and Review of Literature. Oral & Maxillofacial Pathology Journal. 2018 Jan 1;9(1). b) Conclusion preferably to contain a recommendation, your kind suggestion of the paragraph is already included in the paper, so we prefer to keep both.Comment No 5 Ectopic eruption definition needs more justification as the present tooth is ectopic but associated with large maxillary cyst and pushed superiorly as result of cyst enlargementReply to No 5We already put general statement in the introduction about the definition of ectopic eruption and the different theories of the causes of its occurrence were discussed in the discussion part. In our case the third molar is in ectopic location and erupted inside the maxillary sinus where the associated pericoronal cyst was the suggested theory for its location.Comment No 6 Authors did not report any prevalence rate of the dentigerous cyst (which usually associated with impacted wisdom) in the maxillary sinus and also the rate for ectopic impacted teeth in the sinus.Reply to No 6As it is a case report without review of literature, we didn’t go through prevalence of reported conditions, but we agree it is advantageous to be included.Comment No 7 The cystic lesion had discharge and was diagnosed as infected cyst, does the surgeon treat infection before opening the sinus.Reply to No 7It was a chronic condition due to long standing infection, managing the case in this case will be symptomatic only, so we thought that best managing will be through surgical eradication of the source. Possibility of recurrent fistula is high?Patient had a 12 months post-surgery follow-up and no fistula was developed.Why is nasal anticongestant not prescribed as routine post sinus surgery?ReplyIt was prescribed."
}
]
}
] | 1
|
https://f1000research.com/articles/9-209
|
https://f1000research.com/articles/8-1168/v1
|
23 Jul 19
|
{
"type": "Software Tool Article",
"title": "bwimage: A package to describe image patterns in natural structures",
"authors": [
"Carlos Biagolini-Jr.",
"Regina H. Macedo",
"Regina H. Macedo"
],
"abstract": "Currently R is the most popular software for data analyses among biologists. Here, we present bwimage, a package designed to describe patterns from black and white images. The package can be used for a wide range of applications. We implemented functions previously described in the literature to calculate parameters designed originally, but not exclusively, for vegetation structures. Additionally, we propose a new parameter: the aggregation index. We demonstrate applications for field work, providing examples that range from calculation of canopy openness, description of patterns in vertical vegetation structure, to patterns in bird nest structure. We provide advice and illustrated examples of how to produce high quality images for analyses.",
"keywords": [
"animal",
"ecology methods",
"field",
"image analyses",
"image processing",
"vegetation patterns"
],
"content": "Introduction\n\nThe facility to obtain high quality digital images creates the opportunity to measure natural variables using image analyses. Black and white pictures have frequently been used to understand patterns in field ecology, especially in plant biology studies1. However, the use of plant image analyses software is not easily extended to other biological fields for several reasons. Free programs are uncommon and paid software normally has threshold algorithms that were specifically designed for vegetation pictures2. Thus, a flexible method that would allow the application of such analyses to other subjects would be welcome. For example, despite the relatively well reported descriptions of bird nests and egg morphology in Del Hoyo and collaborators3 (but see Xiao, Hu4), there are no well-established approaches to estimate nest wall openness patterns.\n\nCurrently, R software5 allows users to migrate from data processing based on combinations of different software (with the possibility of having costly licensing, software-specific files, incompatibility between operating systems and lack of updates) to a free, single cross-platform software. Here, we introduce bwimage, a package for R that can be used to analyze patterns in black and white images from natural structures. We provide data examples for applications and descriptions of routines for processing of black and white images.\n\n\nMethods\n\nBwimage´s analysis of images is based on the transformation from a picture (“jpeg” and “png” files are allowed) to a binary matrix (Figure 1). For each pixel, the intensity of red, green and blue is averaged and compared to a threshold. If the average intensity is less than the threshold (default is 50%) the pixel will be set as black, otherwise it will be white. Beyond RGB intensity in PNG images, the alpha channel is used to set transparent pixels, i.e. alpha channel values above the threshold (default is 50%) will set the pixel as transparent. In the data matrix, the value one represents black pixels, zero represents white pixels and NA represents transparent pixels. For high resolution files, i.e. numbers of pixels in width and height, we suggest reducing the resolution to create a smaller matrix, as this strongly reduces GPU usage and time necessary to run analyses. However, by reducing resolution, the accuracy of data description will also be lowered. Figure 2 compares different resamplings from a figure of 2500x2500 pixels. If the user is not acquainted with scale and threshold processing and/or images were captured under different light conditions, we recommend the scale and application of threshold algorithms in a native image editor software, such as GIMP6, and subsequent usage of the resulting images with the bwimage package.\n\nA) An image of a natural structure is obtained with digital photography; here we used an image from a canopy. B) The image is converted into a binary matrix, functions threshold_color (to a single image) or threshold_image_list (for two or more images). In the data matrix the value one represents black pixels, zero represents white pixels and NA represents transparent pixels.\n\nA) Original image. B) Black and white conversion of the original image by GIMP software, i.e. all pixels converted to either black or white in image. C–I) conversion of the original image to binary matrices of 10x10, 50x50, 75x75, 100x100, 250x250, and 500x500, respectively.\n\nSeveral metrics can be performed with the functions presented in Table 1. We implemented functions to calculate parameters designed originally, but not exclusively, for vegetation structures (described by Zehm et al. 2003) and propose a new parameter: the aggregation index. The aggregation index is a standardized estimation of the average proportion of same-color pixels around each image pixel. First, the proportion of same-color neighboring pixels (SCNP) is calculated (marginal lines and columns are excluded). Next, the SCNP for all pixels are averaged; then, given the proportion of black and white pixels, number of pixels in height and width, and location of transparent pixels (when present), the maximum and minimum possible aggregation indexes are calculated. Finally, the observed aggregation is standardized to a scale where the minimum possible value is set at zero and the maximum value is set at one (Figure 3).\n\nImagens represent photos of 6x6 pixel, with 50% black and 50% white pixels. For the aggregation index calculation, first, the proportion of same-color neighboring pixels (SCNP) is calculated (marginal lines and columns are excluded). Then, SCNP are averaged. In these examples, the average SCNP is 0.75 (A), 0.5 (B), 0 (C) and 0.875 (D). Next, the maximum and minimum possible aggregation indexes are calculated. In images with 50% black and white pixels, the minimum aggregation will be pixels distributed in chessboard style (C), and the highest aggregation will be the aggregation of all same color pixels on each image side (D). Thus, by scaling the aggregation by the minimum and maximum possible aggregation, the final aggregation index is: 0.857 (A), 0.571 (B), 0 (C) and 1 (D).\n\nBwimage is written in the R programming language5, and can be run on Windows, Mac OS X, and Linux systems. The package is available at the CRAN repository, and the development releases are available at Github (see Software availability)7. The bwimage CRAN page documents package dependencies. Input images must be in one of the following formats PNG, JPG, or JPEG.\n\n\nUse cases\n\nCanopy openness is one of the most essential ecological parameters for a field ecologist. In the bwimage package, canopy openness can be calculated based on a single picture. To illustrate, we demonstrate below how to analyze a canopy image with the bwimage package. The photo was taken with a digital camera placed in the ground, perpendicular to the ground. Canopy closure can be calculated by estimating the total amount of vegetation in the canopy. Canopy openness is equal to one minus the canopy closure. For this example, we used the original image from Figure 1. The original image file is provided as Underlying data8.\n\n\n\nSeveral metrics to describe vertical vegetation complexity can be performed by the bwimage package (see Table 1). Here we provide examples based on an image (Figure 2A) from a vegetation plot of 30×100cm1. The original image file is provided as Underlying data9. On the 100cm side of this plot we placed a panel of 100x100 cm, covered with white cloth, and perpendicular to the ground. A plastic canvas of 50x100cm was used to cover the vegetation along a narrow strip in front of a camera positioned on a tripod at a height of 55 cm. A photograph of the portion of standing vegetation against the white cloth was taken.\n\n\n\nVariation in eggs and nest morphology provide relevant information concerning bird life history that has frequently been used to answer ecological10–12 and evolutionary questions13–15. Here we analyze examples that address the quantification nest wall openness and the aggregation of nest wall holes, using a nest of the blue-black grasssquit (Volatinia jacarina) deposited in the museum collection Coleção Ornitológica Marcelo Bagno, at Universidade de Brasília (register number COMB-N682). Figure 4 describes how to produce a high-quality image to describe patterns in bird nest wall openness. The original image file used is provided as Underlying data16.\n\n\n\nThe following example used a blue-black grasssquit (Volatinia jacarina) nest deposited in the museum collection Coleção Ornitológica Marcelo Bagno, at Universidade de Brasília (catalog number COMB-N682). A) A photo of the nest was taken with a white styrofoam ball (50mm in diameter) placed inside the nest chamber. B) In GIMP software (version 2.10.10), we added alpha channel (to allow transparent pixels), and used the Ellipse select tool to select contrast of the white ball and nest wall, and then erased all background elements. C) To ensure that shadows from nest wall did not influence white ball detection, we also performed the threshold processing in GIMP. To preserve the transparent pixels from background elements, the final image must be saved as a PNG file.\n\n\nConclusions\n\nThe bwimage package provides accessible and simple methods for ecologists and field researchers to describe patterns from black and white digital images. It is a flexible method that allows the application of image analyses to an exceptionally broad range of research subjects. Bwimage´s analysis is based on a simple computational routine based on the transformation of a picture (“jpeg” or “png” files) into a binary matrix, followed by the analysis itself. Several metrics can be calculated by the bwimage package. We implemented functions previously described in the literature, and additionally, we proposed a new parameter: the aggregation index, which generates a standardized estimate of the average proportion of same-color pixels around each image pixel. The application of this methods is exceptionally broad.\n\n\nData availability\n\nFigshare: Canopy of Royal poinciana. https://doi.org/10.6084/m9.figshare.8429117.v28\n\nFigshare: Image from a vegetation plot of 30x100cm. https://doi.org/10.6084/m9.figshare.8429882.v29\n\nFigshare: Blue-black grassquit (Volatinia jacarina) nest. https://doi.org/10.6084/m9.figshare.8432018.v116\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\n- Software available from: https://CRAN.R-project.org/package=bwimage\n\n- Source code available from: https://github.com/biagolini/bwimage\n\n- Archived source code at time of publication: https://doi.org/10.5281/zenodo.32662997\n\n- License: GPL-3",
"appendix": "Grant information\n\nCB received graduate fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe are grateful to Universidade de Brasília for logistic support and Marcelo Antônio de Assis Silva for technical advice on how to obtain a high-quality image of the bird nest used in the manuscript.\n\n\nReferences\n\nZehm A, Nobis M, Schwabe A: Multiparameter analysis of vertical vegetation structure based on digital image processing. Flora. 2003; 198(2): 142–60. Publisher Full Text\n\nNobis M, Hunziker U: Automatic thresholding for hemispherical canopy-photographs based on edge detection. Agr Forest Meteorol. 2005; 128(3–4): 243–50. Publisher Full Text\n\nDel Hoyo J, Elliott A, Sargatal J, et al.: Handbook of the Birds of the World Alive. Barcelona: Lynx Edicions; 2018.\n\nXiao H, Hu Y, Lang Z, et al.: How much do we know about the breeding biology of bird species in the world? J Avian Biol. 2016; 48(4): 513–8. Publisher Full Text\n\nR Core Team: R: a language and environment for statistical computing. 2019.\n\nKimball S, Mattis P, Natterer M, et al.: GIMP Image Manipulation Program. 2019. Reference Source\n\nBiagolini-Jr. C: biagolini/bwimage: Describe Image Patterns in Natural Structures (Version 1.01). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3266299\n\nBiagolini-Jr. C: Canopy of Royal poinciana. 2019. http://www.doi.org/10.6084/m9.figshare.8429117.v2\n\nBiagolini-Jr. C: Image from a vegetation plot of 30x100cm. 2019. http://www.doi.org/10.6084/m9.figshare.8429882.v2\n\nPoláček M, Bartíková M, Hoi H: Intraclutch eggshell colour variation in birds: are females able to identify their eggs individually? PeerJ. 2017; 5: e3707. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGómez J, Ramo C, Troscianko J, et al.: Individual egg camouflage is influenced by microhabitat selection and use of nest materials in ground-nesting birds. Behav Ecol Sociobiol. 2018; 72(9): 142. Publisher Full Text\n\nWang L, Cheng SJ, Hsu YC, et al.: Nest-dismantling behavior of yellow-bellied prinia in mainland and island populations. Acta Ethol. 2018; 21(1): 35–41. Publisher Full Text\n\nLeighton GM: Evolutionary mechanisms maintaining nest construction in avian clades. Avian Biol Res. 2016; 9(1): 44–51. Publisher Full Text\n\nDeeming DC: How does the bird-nest incubation unit work? Avian Biol Res. 2016; 9(2): 103–13. Publisher Full Text\n\nStoddard MC, Yong EH, Akkaynak D, et al.: Avian egg shape: Form, function, and evolution. Science. 2017; 356(6344): 1249–54. PubMed Abstract | Publisher Full Text\n\nBiagolini-Jr. C: Blue-black grassquit (Volatinia jacarina) nest. 2019. http://www.doi.org/10.6084/m9.figshare.8432018.v1"
}
|
[
{
"id": "51562",
"date": "13 Aug 2019",
"name": "Francesco Chianucci",
"expertise": [
"Reviewer Expertise Forestry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is interesting. However, I think the package makes simple things which many other similar packages do, and omit some intermediate steps required to refine the analysis.\nFirst, the authors should provide a list of thresholding (either single or dual) methods to binarize images. The single Otsu, the minimum algorithm, the two-corner method are some of the algorithm which should be developed. The package could also use some dependancies from other packages to implement these thresholding. For instance, the rtiff package contains the function autoThreshold which implement the Ridler-Calvard thresholding. I also suggest take a look at the caiman package which contains tool for canopy image analysis, see also below.\nSecond, users can also process single channel image or decide to use a single channel (for example, the blue channel is frequently used for canopy images). So the user should have flexibility on choosing the image feature to process (setting the channel, setting the threshold).\nCanopy images are usually fish eye images, and therefore they require correction for lens projection. In addition, canopy openness should be weighted for zenith angle in fish eye image. Similarly, gap fraction is required for zenith ring. Thus, the packages should allow to work with fish eye (circular image), namely correct for lens distortion, setting the circular inner mask, extract information for zenith angle ranges (inner rings). I suggest to take a look in the caiman package to inherit its functions.\nAn interesting attribute from the packages is the identification of row or column gaps (holes), namely continuous sequences of white pixels. In my view, a very interesting output would be the identification and the labelling of all holes in the image, along with their summary statistics (e.g. number of holes, size of each hole in number of pixels, average, sd and so on). I think the package will improve strongly if authors can implement such function. This would allow the extraction of canopy attributes from images1.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "4976",
"date": "29 Oct 2019",
"name": "Carlos Biagolini-Jr.",
"role": "Author Response",
"response": "Dear Dr. Francesco Chianucci,We appreciate all of your helpful comments, and we are certain that they have improved significantly the original version of the Package. In the new version (1.2) of bwimage package published in CRAN we: i) introduced two new functions (stretch and compress functions) to provide an additional tool to distort images. These functions are applications of algorithms for mapping images from circle to square, and vice versa, adapted from Lambers (2016 J of Computer Graphics Techniques); and ii) included the possibility of users processing single channel images.We considered incorporating other packages for threshold in bwimage dependencies; however, we decided to avoid a cross-dependency of packages because it implies in a heavy load package, and brings possible compatibility issues with R updates. Thus, we chose to create a tutorial (shared on https://www.r-bloggers.com/using-bwimge-r-package-to-describe-patterns-in-images-of-natural-structures/) to provide an overview of bwimage package and demonstrate examples of how to apply threshold algorithms from the package autothresholdr, following its application in bwimage package. A comparison of estimation of vegetation density from a bush image submitted to different thresholding algorithms is provided. Note: the current version of autothresholdr package (1.3.5) provide 17 threshold algorithms by function auto_thresh, covering a wide range of applications.We do not mention in the previous version of this article, but bwimage version 1.0 already had a function (hole_section_data) to summarize holes statistics (i.e. number of holes, mean hole size, sd, minimum and maximum size). This function was designed to be used inside a loop or apply-family functions. By combination of hole_section_data and loop function, users can potentially collect a summary of holes statistics. The size of each section is obtained by the hole_section function, which returns size and map of each hole. We add information about this function in manuscript table 1. We also provide an example of how to apply hole_section_data for a set of 12 images in the above-mentioned tutorial. With my best regards.Sincerely,Carlos Biagolini-Jr"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1168
|
https://f1000research.com/articles/9-170/v1
|
09 Mar 20
|
{
"type": "Opinion Article",
"title": "Causality assessment of adverse events following immunization: the problem of multifactorial pathology",
"authors": [
"Paolo Bellavite"
],
"abstract": "The analysis of Adverse Events Following Immunization (AEFI) is important in a balanced epidemiological evaluation of vaccines and in the issues related to national vaccine injury compensation programs. If manufacturing defects or vaccine storage and delivering errors are excluded, the majority of adverse reactions to vaccines occur as excessive or biased inflammatory and immune responses. These unwanted phenomena, occasionally severe, are associated with many different endogenous and exogenous factors, which often interact in complex ways. The confirmation or denial of the causal link between an AEFI and vaccination is determined pursuant to WHO guidelines, which propose a four-step analysis and algorithmic diagramming. The evaluation process from the onset considers all possible “other causes” that can explain the AEFI and thus exclude the role of the vaccine. Subsequently, even if there was biological plausibility and temporal compatibility for a causal association between the vaccine and the AEFI, the guidelines ask to look for any possible evidence that the vaccine could not have caused that event. Such an algorithmic method presents some concerns that are discussed here, in the light of the multifactorial nature of the inflammatory and immune pathologies induced by vaccines, including emerging knowledge of genetic susceptibility to adverse effects. It is proposed that the causality assessment could exclude a consistent association of the adverse event with the vaccine only when the presumed \"other cause\" is independent of an interaction with the vaccine. Furthermore, the scientific literature should be viewed not as an exclusion criterion but as a comprehensive analysis of all the evidence for or against the role of the vaccine in causing an adverse reaction. These issues are discussed in relation to the laws that, in some countries, regulate the mandatory vaccinations and the compensation for those who have suffered serious adverse effects.",
"keywords": [
"Vaccination",
"Adverse events following immunization",
"Inflammation",
"Autoimmunity",
"Genetic susceptibility",
"Multifactorial diseases",
"Mandatory vaccinations",
"Injury compensation"
],
"content": "Introduction\n\nPublic health policy supports broad vaccination and, at the same time, acknowledges the prospect of adverse events following immunization (AEFI), harming a few “unlucky” individuals. Most countries have introduced laws that allow compensation for people who think they have been seriously and/or permanently damaged by recommended or mandatory vaccines, or for families in case of death. An AEFI is defined as “any untoward medical occurrence which follows immunization and which does not necessarily have a causal relationship with the administration of the vaccine. The adverse event may be any unfavorable or unintended indication, abnormal laboratory finding, a symptom or a disease.\"1. Although the rules introduced by different countries often diverge2–4, an essential part of the evaluation of AEFI is the search for whether or not there is a causal link between the administered vaccine and the subsequent pathological phenomenon. It is evident that in this area, the causality assessment plays a crucial role for both public health policies and any possibly injured individuals.\n\nWHO guidelines for causality assessment1, provide that “Allegations that vaccines/vaccination causes adverse events must be dealt with rapidly and effectively. Failure to do so can undermine confidence in a vaccine and ultimately have dramatic consequences for immunization coverage and disease incidence long after proof is generated that the adverse event was not caused by a vaccine (e.g. autism and MMR, encephalopathy and pertussis).” This articulation is entirely understandable and plausible. The same guidelines do not mention the affected individual for whom a causal inquiry is important, as in the absence of damage causation recognition, the harmed individual cannot access any compensation by provided programs. In addition to generating an obvious injustice, too rigid and restrictive rules could undermine the confidence of the population in the vaccine solution, create an expectation of claim denial and lead paradoxically to a decline in coverage. Present trends in several countries towards an increasing number of mandatory vaccines, is a delicate and controversial subject, impacting both social and economic concerns5. This is another reason why it is important that the procedure of causality assessment is accurate in theory and practice.\n\nCausality is the relationship between two events (the cause and effect), wherein the second event is a consequence of the first. The WHO guidelines1 acknowledge that “Sometimes there are multiple factors that may precipitate the effect (event) or may function as co-factors so the side effect (event) occurs.” As far as vaccines are concerned, the fact that severe reactions affect only a few individuals suggests in most cases vaccines are not the only cause of the event and further factors are necessary in the development of pathology.\n\nThe growth of multifactorial diseases in the last decades has led to the development of the “medicine of complexity\"6, ranging from cardiology7 to epidemiology8,9, from pharmacology10 to nursing care11, or forensic medicine12. To underline the importance of the topic in modern medicine, in 2002 the journal Science dedicated a whole issue (vol. 296, n. 5568) to the “puzzle of complex diseases”, including papers on the causes of diabetes13, systemic lupus erythematosus (SLE)14, schizophrenia15, and considering the challenges of sorting out the multiple genetic, infectious and life-style factors and their interaction in the pathogenesis of common diseases. The pathogenesis of autoimmune disease is characterized by a complex interaction between genetic and environmental factors, and immune and hormonal reactions, which is the much talked of “mosaic of autoimmunity\"16. In vaccinology, the new developing fields of “vaccinomics” and “adversomics” exploit the powerful tools of bioinformatics to study adverse side effects to vaccines, using a systems biology approach17–22. Furthermore, disorders characterized by episodes of exaggerated inflammatory response “hyperinflammatory states” or “autoinflammatory syndromes” develop as multifactorial diseases, affecting the severity and frequency of clinical findings23,24.\n\nTo ensure compliance with the above criteria and wider acceptance of the results, the WHO recommends that the assessment of AEFI causality is performed by a multidisciplinary committee comprised of experts from paediatrics, neurology, general medicine, forensic medicine, pathology, microbiology, immunology and epidemiology. In this opinion article, the problem is addressed from the standpoint of general pathology and immunopathology. In order to frame the correct perspective and scientifically founded causation assessment, it is appropriate to summarize the main mechanisms of vaccines and the possible reasons for a severe adverse reaction. This knowledge is essential in order to properly utilize the WHO algorithm, where the plausibility and temporal compatibility of an AEFI is evaluated.\n\n\nThe complexity of reactions to vaccines\n\nVaccines are mixtures of substances that cause milder forms of diseases, or mimic those of real diseases and, therefore can cause harm. The latest publication of the Italian Drug Agency (AIFA) July 30, 2019 (https://www.aifa.gov.it/), reports that serious AEFI correlated to vaccines in 2018 were 3.1 per 100,000 doses, with considerable differences between the different vaccines, for example the vaccine MPRV correlated with a rate of 12.7 reports per 100,000 doses. On the other hand, there is evidence of significant differences in rates of AEFI, according to the methods of data collection. A recent paper reported a notification rate of 3,800 correlated adverse events per 100,000 doses of measles/mumps/rubella/varicella (MMRV) vaccine (often administered together with anti-hepatitis A)25. The latter publication states that the use of “active” reports data is essential for the study of adverse events defined as “rare\" (those whose prevalence is less than 1/1,000 doses).\n\nA vaccine may cause serious adverse reactions for three reasons: a) the material, that is, the content is “defective” or “contaminated\", due to preparation or storage inaccuracies; b) administration errors, such as accidental intravenous injection, the injection near a nerve plexus or the delivery of aluminium in the skin instead of into muscle; c) abnormal “reaction\", manifesting excessive biological stress caused by a foreign material or live attenuated virus. The most severe reactions are related to the vaccine action that involves two types, an “innate immune response\", linked to the early biological defences to injected matter, and a more antigen-specific response, linked to the adaptive immune defences (Figure 1).\n\nLeft column: normal responses; right column: possible pathology (excess/disorder of biological responses). Image is author’s own, produced for this review.\n\nThe first phase immediately following the inoculation of the foreign material is the activation of a local inflammatory reaction at the point of injection, primarily involving the phagocytic cells such as monocytes and macrophages; the inflammation certainly leads to the production of cytokines by epithelial, mesenchymal and nerve cells: the release of classical inflammatory cytokines such as IL-1, IL-6, TNF-alpha, occurs a few hours after injection of aluminium-adjuvanted vaccines26. When the reaction is sufficiently strong, the local inflammatory mediators (complement, cytokines, chemotactic factors) spread and amplify the reaction at a systemic level, which explains the general and neurological symptoms in the first hours or days after the vaccine. There appears to be a strong relationship between increased concentration of cytokines and febrile reactions, lymphadenopathy and generalized rash, after yellow fever vaccination27–29.\n\nIn very general terms, the inflammatory phase becomes pathological when it is in “excess\", i.e. causes negative side effects that outweigh those necessary to achieve the protective and repairing purposes. As for vaccines, fever is a useful mechanism to promote the mobilization of cellular, vascular and metabolic defences, to kill viruses and activate immunity, but it becomes “disease” beyond a certain temperature (the so-called “hyperthermia”). Conventionally, hyperthermia superior at or in excess of 39.5°C is considered a severe adverse reaction to the vaccine, and may cause seizures. The risk of febrile seizure increases over 5 times in children aged 12 to 35 months, within 6 to 11 days after exposure to the measles/mumps/rubella (MMR) vaccine30,31. Compared with MMR alone, the MMRV vaccine doubles the risk of febrile seizures in children aged 10–24 months, and does not modify it in children between 4 and 6 years32. Receipt of DTP vaccine, but not of DTaP33, was associated with a 5-times increased risk of febrile seizures on the day of vaccination34.\n\nFebrile seizures represent a predominantly functional disorder and are generally considered relatively benign, i.e. do not leave organic brain damage. However, in the case of longer term high temperature and convulsions (complex seizures, defined as an episode >15 min or recurrence within 24 hours35), or in patients with cardiovascular system diseases, the brain can suffer from disturbance even on the level of cell viability, related to inflammation itself (cell damage due to excitotoxicity or to oxygen metabolites secretion by microglia), respiratory distress and anoxia. In extreme cases, prolonged hyperpyretic seizure syndrome can result in brain degeneration and/or death36–40. As for the long-term consequences, the risk of developing epilepsy after complex febrile seizures is estimated at around 10–20%41.\n\nA particular role may involve simultaneous injections of vaccines, because it is obvious that the type of nonspecific reactions are enhanced, if they are due to additional pathogenic factors26. This is even more evident when you consider that vaccination is generally not recommended in children who have a febrile condition, whatever the cause.\n\nThe second step of the vaccine function is the activation of immune system through antigen presentation by mononuclear phagocytes to lymphocytes. At this stage, a pathologic reaction may consist of unwanted responses due to hyper-immunization, autoimmunity, allergy and damaging infection (the latter in case of live viruses in immunocompromised patients).\n\nHyper-immunization reactions for repetitions of the tetanus vaccine have been documented in the Italian population, where the prevalence of an excess of antibodies (>5 IU / ml) was described in 17% of the observed subjects42. The diseases induced by hyper-immunization following the administration of vaccines are due to sensitivity to one of the components of the vaccine, and exacerbation of atopic or vasculitis symptoms43,44. If the status of pre-existing immunity is unknown, to avoid hyper-immunization and its risks, it is recommended to carry out laboratory testing to determine antibody titre and avoid vaccination if the titre is already high enough45.\n\nVaccines have long been suspected of playing a negative role in inducing autoimmune diseases46–56. The most established connections between autoimmune disease and vaccinations have been reviewed57 and include: immune thrombocytopenia after MMR vaccination, Guillain–Barré syndrome after swine influenza vaccination, reactive arthritis after hepatitis B and rabies vaccinations, SLE and other autoimmune diseases, after hepatitis B and human papilloma virus vaccinations.\n\nEvaluation of the association of AEFI with autoimmune diseases is challenging due the complex innate and adaptive immune responses to vaccine components (adjuvants, antigens, preservatives) that may contribute to reactogenic responses17. The specific components of the vaccines (antigens) can trigger immunity against microbial antigens, but also a self-immunity in the case of there being a molecular mimicry (similarity) between antigen protein sequences and protein sequences of components of the organism or HLA receptors16,52,58–60. The emergence of the post-vaccination autoimmune syndrome is associated with genetic predisposition, for example, HLA-DRB1 or HLA-DRB4, as a result of exposure to additional external factors or endogenous autoimmunity triggers61–63.\n\nA typical autoimmune disease, which seems to be related to immunization in about a third of cases64,65, is SLE (Figure 2). SLE pathogenesis is very complex, subject to both environmental factors – like viruses66, bacteria67, but also to drugs68 (and this is highly significant) – and also to genetic susceptibility (for example HLA polymorphism)69,70 as well as hormonal factors (in fact, it has a considerable prevalence in the female gender)71. Moreover, the vaccine adjuvants can increase the immunogenicity of the injected antigens and, as a consequence, may also increase the risk of triggering autoimmune adverse events56.\n\nThe image of the body is by Mikael Häggström, used with permission (in the public domain).\n\nAccording to some authors, it may be possible to conceive the production of vaccines based solely on unique sequences of pathogens, which could then frustrate the potential risk of cross-reactivity in the existing vaccine formulations72,73. Unfortunately, this approach is still utopian for vaccines currently in use.\n\nBoth local inflammatory responses and immune systemic responses are increased by aluminium adjuvants. Concerns about the safety of aluminium emerged as a result of the recognition of its biological persistence, exhibiting an unexpectedly long duration within immune and nervous system cells74–82. Aluminium adjuvant particles remain in the lymphoid organs and can even get into the brain, a phenomenon documented in animal models83,84. In particular, the long-term persistence of an aluminium granuloma, also defined as macrophagic myofasciitis, is characterized by chronic arthromyalgia, fatigue and cognitive dysfunction74,85. The problem is not just a difficulty in curing local granulomatous inflammation86, but the systemic effects, such as oxidative stress on the blood87, cognitive dysfunctions88,89, chronic fatigue syndrome90,91, hypotonia92, child motor retardation93, sensory disturbances, loss of vision and cerebellar signs94, as well as alterations of cerebral circulation95. A recent review illustrates several mechanisms by which aluminium introduced through immunizations can produce chronic neuropathology in genetically susceptible individuals96. These authors recommend that the use of aluminium salts in vaccines be discontinued in favour of adjuvants less involved in the activation of autoinflammatory and neurolesive reactions.\n\nThe mere fact that vaccines are followed by serious adverse effects in only a few individuals suggests that in most of these cases underlying susceptibility factors are present, which predispose or prepare the complex innate or adaptive reaction system for an excessive or biased reaction. Among these factors, it is obvious that genetically defined pre-existing conditions are at work in some groups of subjects in the population18, but research in this field is in its infancy. A genetic predisposition results from specific genetic variations that are often inherited from a parent. These genetic changes contribute to the development of a disease but do not directly cause it.\n\nThe genetic conditions that have been associated with excessive vaccine reactions can be various (Table 1), as congenital immunodeficiency, variants to the virus receptors and cytokines, epileptic tendencies, defects of detoxification inhibitors and enzymes, and so on. However, the small size of the groups, the often anecdotal type of the reports, and the multifactorial nature of diseases, do not allow us to draw definitive conclusions on causality regarding the observed associations between genetic types, vaccine and reported outcomes.\n\nAGG: agammaglobulinaemia; CID: combined immunodeficiency; CVID: common variable immunodeficiency; HGG: hypogammaglobulinaemia; SCID: severe combined immunodeficiency; MBL: mannose-binding lectin; TLR: toll-like receptor; MTHFR: 5,10-methylenetetrahydrofolate reductase; IRF1: interferon regulatory factor-1; SCN1A: sodium channel, voltage-gated, type I, alpha subunit; PCDH19: protocadherin 19; P2X7 is a purine (ATP) receptor; IL17R: interleukin-17 receptor; PI3K: phosphatidylinositol-3-kinase; AS03: adjuvant systems 03 (oil-in-water emulsion); GDNF-AS1: glial-derived neurotrophic factor antisense RNA-1; GSD: glycogen-storage disease.\n\nFebrile seizures are genetically complex disorders, believed to be influenced by variations in several susceptibility genes129 and, among the susceptibility genes, by those encoding cytokines of the acute phase130. The risk of post-vaccination febrile seizures increased in subjects with previous and family history of febrile convulsions, showing that in some subjects there is a predisposition, which is obviously a co-factor in determining the risk of the vaccine131,132. The latter authors suggested that in order to reduce the risk of adverse reactions to MMRV, children with a family history of febrile seizures should not be vaccinated. Two loci were clearly associated with febrile seizures MMR-related, but not with those from other causes118: the IFI44L interferon-stimulated gene and the CD46 receptor for measles virus. It is interesting that the same IFI44L and CD46 genes are among those that affect the magnitude of the antibody response to measles133.\n\nAmong the genetic factors and in particular polymorphisms, the role of SCN1A gene mutations is demonstrated by the fact that the risk of post-vaccine seizures increased in patients with Dravet syndrome, a severe epileptic encephalopathy114,116,134. Vaccination is the trigger of the first seizure in about 50% of cases135, while the vaccination program does not seem to increase long-term consequences of the disease on cognitive function. In children, genetic or structural defects are an underlying cause of epileptic seizures onset, after routine immunization, that may act as a triggering factor136 and these authors suggested that early genetic testing should be considered in all children with vaccination-related onset of epilepsy. In this context, it is important to point out that live vaccine may be safely administered to children with Di George syndrome, a congenital T-cell defect associated with a deletion in chromosome 22 (22q11. 2 deletion)137.\n\nAs a first step towards a systematic collection of genetic susceptibility factors, Lin, He and Xie have created an ontological framework (Ontology of Genetic Susceptibility Factors, OGSF), which may provide guidance for representing diverse types of genetic susceptibility factors for vaccine adverse events, such as HLA alleles, SNPs, genes, and gene haplotypes138–140.\n\nIn the complex mechanisms regulating the innate and adaptive responses to immunization, the role of the microbiome and of the intestinal barrier should be noted as important factors that may contribute to systemic inflammatory reactions141–143. Bacterial endotoxins that may be released from the intestine, in the case of increased intestinal permeability (also possibly caused by medication or dysmicrobism), should not be neglected. The interactions between products of the bacterial microbiome with immune cells trigger self-reactivity, chronic inflammation and tissue damage in genetically sensitive subjects144,145. The synergy between LPS and inflammatory cytokines is one of the simplest and most ubiquitous mechanisms of neuroinflammation and neurodegeneration143,146–148. This can happen through the modification of substances by the intestinal bacterial flora, which can therefore become autoantigens and mistakenly trigger immune responses of the wall itself. In addition, recent studies have shown that a breakdown or increase in permeability of the intestinal barrier and the translocation of commensal bacteria or endotoxins into non-intestinal organs can trigger several autoimmune pathways141–143,149. For example, many people with multiple sclerosis have been shown to have an altered microbiome, increased intestinal permeability and changes in bile acid metabolism150. Allergic diseases and autoimmune encephalitis have also been correlated with changes in intestinal microbiota151. For these reasons and as a precautionary measure, the healthy state of the gut should always be considered, before a vaccination procedure.\n\n\nWHO guidelines\n\nWHO guidelines for causality assessment were published in 2013152 and updated in 20181 (https://www.who.int/vaccine_safety/publications/gvs_aefi/en/).The first step is to determine if the AEFI is “eligible”, meeting the minimum criteria for the assessment of causality, such as the presence of a clear diagnosis. The second phase (\"checklist\") encompasses a systematic review of relevant and available information to deal with possible causal aspects of the AEFI. Then, an “algorithm” synthesizes the entire conceptual and methodological process in four steps (Figure 3). Given the central importance of the WHO algorithm, for brevity and clarity of need, this article will focus on these steps, with four notes highlighting the main problems that emerge in the light of the previous discussion on the possible mechanisms of vaccination adverse reactions.\n\nThe WHO algorithm of step 1 rules out the association of an AEFI with vaccination if there is another cause. This is the first and decisive criterion for exclusion and is stated on the “checklist”, alongside the question “Is there strong evidence for other causes?” The text then further explains that a detailed medical history, clinical examinations and investigations, including laboratory tests on the patient, can help identify other conditions such as other diseases and congenital anomalies that may have caused the event. Provided is the example of the death of a girl, following vaccination against human papilloma virus (HPV), where a post-mortem examination accredited the cause to a malignant mediastinal tumour.\n\nIt should be noted that, in this first phase, the process systematically seeks another “strong” cause which, if found, would exclude the causal link. This concept of “strength” is not defined and can be misunderstood. According to medical historian Cosmacini153, causality criteria are changing due to epidemiology of modern diseases: a strong causality criterion arises when the pathogenic cause, for example, the infectious agent, is “forcibly” followed by pathological effect, i.e. the disease or event; while a weak causality is when the cause has “less strength” or “relative weakness”. In these second possibilities, the relative weakness lies in the fact that the pathogenic cause or causes are probabilistically followed by the morbid effects. As explained above, in the presence of many possible causes, none of which is necessary or sufficient to determine that disease, the traditional concept of cause is transformed into the new concept of “risk factor.” The multifactorial perspective, deriving from the observation of adverse reactions from the vaccine and from the recent knowledge of pathology and immunopathology (see above sections), means that the concept of “strength” of causal association takes on a probabilistic meaning.\n\nThis is a very important aspect and should not be overlooked. If a disease is multifactorial in its nature, to be considered strong enough to exclude the contribution of a vaccine, the “other cause” must be independent of a possible synergistic interaction with the effects of the vaccine itself on the immune responses. For example, a complication of an advanced tumour, after pneumococcal vaccination can be considered “the” cause of death (as in the example provided by WHO guidelines), but a HLA haplotype that predisposes to autoimmunity cannot be considered “the” cause of an autoimmune disease that arose after the hepatitis vaccination.\n\nThe second step of the algorithm (Figure 3) includes the evaluation of the “positive” elements that could be in favour of a consistent causal relationship, considering the biological plausibility and whether the event occurred inside a time window compatible with the risk after vaccination. If all these aspects are in favour of a role of the vaccine in the AEFI, without negative evidence, the causal association is classified as “consistent”. The biological plausibility of damage from the vaccine can be inferred by its known action: in some rare and susceptible individuals, an abnormal response to the stimulus, provided by antigens and adjuvants, unfortunately occurs. The person who has suffered damage from the vaccine usually was predisposed via some “risk” factor – genetic or acquired - which was not sufficiently “strong” to cause illness or injury. With vaccination moreover, the organism is destabilized leading to a pathological reaction. From this point of view, a steadier plausibility of a consistent association between disease and vaccine is generated, if two conditions occur: a) the same pathology (or a biologically similar one) has already been described in other cases after vaccination and b) the subject is a carrier of an increased proneness to that particular pathology.\n\nConcerning the “time window” in which an AEFI can be considered as putatively associated with vaccination (box II in Figure 3), the questions are usually clear enough and should not pose significant issues, at least when previously described cases exist and for the “acute” cases (hours or days after vaccination). However, problems can be substantial when considering chronic illness and autoimmune disease, which can develop quite some time after vaccination. For autoimmune diseases, the difficulty is even greater because, as we have seen, they are “weakly” associated with vaccination. For example, a systematic review and meta-analysis suggests that vaccinations significantly increase the risk of SLE (RR=1.50; 95%CI 1. 05-2. 12, P=0. 02) and rheumatoid arthritis (RR=1. 32; 95%CI 1. 09-1. 60, P=0. 004)65. This means, from a theoretical point of view, that about one in three SLE cases may also occur via the vaccine. However, among all people with SLE, we cannot say for whom the vaccine has had a causal role; we can only say, for a person who has had the appearance of SLE after vaccination, that there is about a 3 in 10 chance that vaccination has contributed negatively to the development of the disease.\n\nIt should be noted that eight patients with disseminated acute encephalomyelitis occurred less than 10 weeks after vaccination against hepatitis B (HBV) have been reported154. Another case, which occurred 3 weeks after HBV vaccination, has been described more recently155 and 93 patients with autoimmune disease after HBV vaccination have been reported47. The mean latency period since the last dose of vaccine and the onset of symptoms was 43 days. However, studies of cohorts of children with neurological and behavioural syndromes (e.g. anorexia nervosa, attention deficit hyperactivity disorder, obsessive-compulsive disorder) have observed an increased risk of up to 12 months after vaccination156. On the other hand, given the multifactorial nature of most chronic diseases, it cannot be excluded that, over a longer period, other causes determining the chronic pathology, unrelated to the vaccine, may well have occurred. Consequently, in the perspective of a single case, it is very probable that the undesired reactions which arose at a considerable distance from the vaccination escape the possibility of proving a causal association.\n\nIncidentally, it should be noted that the WHO algorithm published in 2018 (Figure 3) lacks the indication “No” over the arrow connecting phase II box “Was the event within the time window of the risk?” with the phase III box “Is there a strong evidence against a causal association?”. As a matter of fact, in the algorithm of causation assessment published in 2013 there did appear the inscription “No” on the connection arrow157,158. This omission in the recent algorithmic form can create misunderstandings, because those who follow the algorithm literally would be prevented from concluding with IIA (\"Consistent causal association\") and would necessarily proceed to phase III, a passage where again an exclusion criterion is offered, mostly linked to literature (see below). Thus the path proposed by the arrow would defeat any chance of reaching the IIA conclusion, although other evidence is favourable to a positive causation. To avoid any such misunderstanding, it would be more correct and reasonable to restore the previous descriptor “No” to the aforementioned connection.\n\nIn phase III of the algorithm, users must answer these key-questions “Is there strong evidence against a causal association?” and “Is there a body of published evidence (systematic reviews, GACVS reviews, Cochrane reviews, etc.) against a causal association between the vaccine and the event?” This is a criterion used in a very “strong” way, even to the point of excluding a case for lack of evidence of literature, leading to conclusion IIIA (“Inconsistent causal association”), even if there is plausibility for a consistent association and a compatible time. The topics reported as emblematic are autism and sudden infant death syndrome (SIDS), as it is argued that according to the literature they cannot be caused by the vaccine. Specifically, the guidelines1 write that “no evidence exists of a causal association between MMR vaccine and autism or autistic disorders” and that “the committee concluded that vaccines did not cause SIDS.” Here we must pay attention to language and related concepts, because the “lack of evidence of association” may become easily, but mistakenly, “evidence of the lack of association”. It would not be correct to use this lack of knowledge as a “guillotine” criterion to exclude causation in individual patients. In fact, epidemiological studies cited in the document may exclude an association at the population level but do not have the power to exclude rare cases, especially if the surveillance is not 100% efficient.\n\nIn a Cochrane review of 201230 the design and reporting of safety outcomes in MMR vaccine studies, both pre- and post-marketing, are defined as “largely inadequate”. The question is how one can exclude any liability of the vaccine in contributing to the development of serious neurologic adverse effects, at least in sporadic cases of children predisposed via other genetic factors. The genetic background of autism is known (about a quarter of cases of autism have a genetic basis although only in rare cases is the disease totally genetic) but encephalopathy may also be determined by autoimmunity159–163, which, in turn, depends on some factor of an antigenic nature. It should also be noted that “autism” is not a disease with specific symptoms and reproducible in all subjects with personality disorders, so that we speak of “autism spectrum disorders”. The most obvious case of a possible overlap between autism spectrum symptoms and another disease, surely caused by vaccine adjuvants, is the macrophagic myofasciitis164–167.\n\nRegarding SIDS (otherwise known as SUDC, sudden unexplained death in childhood), the considerations are partly different. SIDS (or SUDC) is strictly defined as “unexplained crib death from known causes upon autopsy”38. The prevailing literature, as rightly reported by WHO, states there is no association between SIDS, SUDC and vaccination. This is completely obvious, because if in a case of “death in a cradle” there is no symptom of particular diseases, nor any autopsy findings that can highlight the cause, this means that the role of the vaccine cannot be proved either. On the other hand, what the WHO algorithm can’t exclude is a violent adverse reaction to the vaccine components that can result in death in particularly fragile subjects. This type of reaction would not correspond to the definition of “SIDS” or “SUDC”. For this reason, in the case of a sudden death of a child after vaccination, before adopting the exclusion criteria of Phase III, the analysis of the case should exclude any clinical evidence (e.g. high fever, convulsions, respiratory distress, syncope) and autopsy finding (e.g. cerebral congestion, pneumonia, isolation of vaccine virus strain, significant increase of some cytokines in the blood) of strong inflammatory reactions. It was reported that in six cases of children who died in the crib after hexavalent vaccine and were previously diagnosed as “SIDS”, the autopsy revealed severe signs of encephalitis and other laboratory data indicating systemic inflammation168. The literature does not exclude the fact that pneumonia is rarely as a result of vaccination, since cases of the syndrome have been reported in deceased patients after DPT vaccination169, influenza170, and anti-haemophilus influenzae type b171. In summary, the benchmark linked to the scientific literature is important for the final categorisation (step IV), but should not be considered as a decisive criterion leading to conclusion IIIa, i.e. to exclude causal association in individual cases.\n\nEventually, the evaluation process ends with a global assessment, according to four categories: “consistent” when the causal association between the event and the vaccine is considered plausible, “inconsistent” in the presence of other causes which can justify the event, “indeterminate” when the evidence is insufficient to support a causal relationship in the presence of confounding factors, and “unclassifiable” when the information necessary to carry out the assessment is inadequate.\n\nApart for the “unclassifiable” cases, for which there is no possible classification, the other categories must be discussed and weighed carefully. The distinction between “consistent” and “inconsistent” could possibly apply to some clear-cut cases, but it becomes forced when you see the adverse reaction to the vaccine manifesting itself as a complex and multifactorial process, wherein the predisposing conditions and the trigger are contributory causes, with different pathogenic mechanisms. The perspective adopted in this report implies that although different causes contribute to an adverse event following immunization, they cannot be considered necessary and sufficient “causes” of the event per se except in very special cases. This problem is not just a difficulty of language and definition, but reveals the conceptual approach adopted by WHO in the preparation of the guidelines in question, under which one proceeds “by exclusion”, in search of an “other cause “. But if the abnormal reaction to the vaccine (which has already been made in the diagnosis) has a multifactorial origin, proceeding by elimination of one or more con-causes is incorrect from a scientific perspective. For example, if a child affected by a serious heart condition, dies the day after vaccination, which led to strong fever and/or difficulty breathing, the most plausible hypothesis is that the effect was determined by the “cooperation” of two factors, both important and interacting, but none of which alone could explain the event, without the other. This point was already raised by Puliyel, Naik and Phadke who noted that, according to the WHO algorithm, a cardiac decompensation in children with an underlying heart disease “would not be considered causally related to the vaccine, although vaccination contributed to cardiac failure”158,172.\n\nA problematic approach to the causal assessment appears where the guidelines1 state that “In doing causality assessment on an individual case report, it must be remembered that in essence one is conducting a differential diagnosis\" (page 7) and that “it is important to recognize that causality assessment of an AEFI in an individual patient is an exercise in medical differential diagnosis. A good clinician does not diagnose diabetes or coronary artery disease on the basis of conflicting or vague information. In the same way, an AEFI should not be causally linked to a vaccine without adequate information\" (page 34). Giving examples of such “differential diagnosis” can be misleading, because normal clinical activity is very different from causality assessment. In fact, the “differential diagnosis” of a multifactorial disease normally is not based on the cause but on its clinical manifestation, that is, the signs and symptoms, the pathological findings, and laboratory results. On the other hand, in the case of an AEFI one is not conducting a “diagnosis” of the disease (also here the WHO procedure provides for a diagnosis be made from the beginning, otherwise the case is not even “eligible”), but is trying to determine what was the sole cause or were the plural causes of the reported adverse event.\n\nTo illustrate how the application of the WHO algorithm is difficult and potentially error-prone, three case studies are presented (Box 1) in which the death of children occurred within a short period of time after vaccination. These cases are described in the AIFA reports of vaccine surveillance of AEFI relating to 2016, 2017 and 2018 years (https://www.aifa.gov.it/rapporto-vaccini). In all reported cases the causality link with the vaccine was excluded because of the presence of “other causes”. These examples raise some questions and deserve clarification, without which a high risk of misinterpretation exists. The notes of the author concern a) whether the alternative “other cause” was sufficiently clear and “strong” as a diagnosis and as a possible cause of death and b) whether or not there could be a plausible interaction between the pre-existing clinical conditions and the biological action of the vaccine.\n\nCase 1:\n\nCase cited in AIFA 2017 Report for 2016: “Preterm baby girl (born after 34 weeks growth in utero), vaccinated at 11 weeks with Infanrix xexa, Prevenar 13 and Rotarix. (. . .). The death occurred about 20 hours after vaccination, due to sudden death classified by the whistle-blower as “death in a cradle”. The autopsy study revealed signs of pulmonary and meningeal congestion and a finding of liver vacuolization compatible with lipid metabolism disease. The causal link was not correlated with vaccination, due to the detection of another possible known cause of death (congenital defect of lipid metabolism) [references 1–3].\"\n\nIn this case, the role of another possible known cause of death seems to be clearly described: the autopsy finding of “liver vacuolization” as “compatible” with the lipid metabolism disorder, but it is certainly not an accurate diagnosis. Given that the autopsy showed “lung congestion and meningitis,” how does this finding connect with an inborn error of lipid metabolism, which until then had presented no clear symptoms and was never diagnosed? To justify the possible “death in crib”, the genetic variant apoEe4174, is then cited in bibliography no. 3 leaving it to be understood that this defect could be the cause of death in the cradle. However, given a careful reading of the work, apoEe4 has equal prevalence in children with SIDS and healthy children. Furthermore, apoEe4 does not appear to cause liver vacuolization. Finally, in the hypothesis that the girl had a disease of lipid metabolism, how is it excluded that 3 different vaccines injected simultaneously may have been the trigger, since signs of lung and meningeal congestion were detected (more compatible with systemic inflammation than with lipid metabolism disease)? It is known that respiratory distress is described as an adverse reaction to hexavalent and encephalitis (remember meningeal congestion) and in rare cases has been associated with vaccination113,175. It should be remembered, moreover, the vaccine data sheet hexavalent provides that “when Infanrix hexa is co-administered with a pneumococcal conjugate vaccine or with the vaccine MPRV, the rate of febrile reactions is higher in comparison to what occurs as a result the administration of Infanrix hexa alone “ and that on the data sheet of the vaccine Rotarix, it is indicated that vaccination “should be postponed in babies, who have a sudden high fever, diarrhoea or vomiting. “ In brief, the question is whether or not, in a case like this, the “other cause” is “strong” enough to exclude the possible pathogenic effects of the vaccine, when the autopsy and the time window are compatible.\n\nCase 2:\n\nCase cited in AIFA Reports for 2016 and 2017: “20-month-old infant vaccinated with Neisvac-C. Two days after the administration of Neisvac C, reported feverish rise followed by death after a few hours. The whistle-blower reports that, at the time of the sanitary intervention, it was only possible to ascertain death. The death was diagnosed as “Sudden Unexplained Death in Childhood, SUDC” as a result of “hyperpyretic hyporeactivity” in the course of respiratory infection with viral aetiology and body temperature at the time of death at 41°C, arisen 52/53 hours after vaccination. Considering this evaluation, the causal link is “not related” to vaccination due to the simultaneous presence of another cause. ”\n\nThe SUDC is by definition “the sudden and unexpected death of more than one year old postnatal child that remains unexplained after a review of the medical history, the circumstances of the death and a complete autopsy”38. So, how this “diagnosis” of SUDC is to be reconciled with “respiratory infection with viral aetiology,” remains to be clarified. Either it is respiratory infection, or a SUDC. Moreover, the attribution of the pathology to a viral aetiology remains unexplained, given that no virus was isolated and that “at the time of the intervention of the health workers, it was only possible to ascertain death”. Finally, the “hyperpyretic hyporeactivity” as a possible cause of SUDC is totally speculative, given that this condition/diagnosis/symptom is not described in any scientific literature. In this case, there is biological plausibility and a time window compatible with a pathogenic role of the vaccine in the triggering of an extremely strong inflammatory systemic reaction: a strong fever is a very common consequence (1–7% of cases) of vaccination with Neisvac-C and which can occur in the first 6 days176. Why was the causal link excluded a priori without considering the vaccine at least as a “contributory” cause?\n\nCase 3:\n\nCase reported succinctly in the report AIFA 2018: Serious adverse reaction to hexavalent plus pneumococcal vaccines, where death was found “not related on the basis of available information”: 6-month-old male patient with Down syndrome and congenital heart disease diagnosed as Fallot tetralogy associated with a complete atrioventricular septal defect-Rastelli type A, already subject to hypoxic crises in relation to paraphysiological stimuli”.\n\nDown syndrome itself does not cause death; heart disease is certainly a potentially fatal condition but often has a chronic course so that it has also been treated surgically, even in a patient with Down syndrome177. However, in this case there is no certainty about the cause of death, i.e. evidence that the congenital malformation resulted in death on that occasion, independently of the vaccine delivered right before. No autopsy evidence is reported from which it can be understood if it was a hypoxic crisis, or signs of systemic, pulmonary or if cerebral inflammation had been detected. To rule out the role of the vaccine, in a case like this it would be important to know if the child had developed a fever, also in light of the fact that two vaccines were administered simultaneously (see note on Case 1). The problem is significant because if there had been systemic inflammation (reported by fever or other serum laboratory findings), the biological plausibility of the interaction with the vaccine would exist. In a child who certainly has a strong underlying pathophysiological fragility (\"hypoxic crises in relation to paraphysiological stimuli\"), how can it be excluded that simultaneous injection of hexavalent and pneumococcal vaccines (both of which are known to cause adverse reactions with respiratory diseases and therefore hypoxic crisis) may have contributed to the cardiovascular arrest? In a case like this, the “other cause” of death seems “strong”, but it is not “independent” of a possible triggering effect of the double vaccine.\n\n*https://www.aifa.gov.it/rapporto-vaccini. The translation from the original Italian text is by the Author.\n\n\nConcluding remarks\n\nAlthough all licensed vaccines are generally safe for the majority of people, the vaccinated may still suffer adverse events in reaction to various vaccines, a few of which can be serious or even fatal140,173. Regarding public health, the proper identification and classification of AEFIs allows for the most accurate information possible about the true frequency of certain ailments in combination with vaccines, thereby minimizing vaccine risks, reassuring the population and informing national or global public health strategies. Regarding the damaged individual, a sound causality assessment supports the affected individual (or family) pursuing any compensation scheme(s), if a consistent association of his/her illness with vaccination is demonstrated. Italian law, n. 210/1992, provides for such compensation in the form of a monthly allowance from the State to any person who has been, due to mandatory (or recommended) vaccination, the subject of injury or illness from which is derived a permanent impairment of physical and psychological integrity. In Italy, 691 people have been recognized as permanently damaged by vaccinations, including 27 deaths (http://www. condav.it/).\n\nThis article has described the complexity and variety of adverse reactions to vaccines, from the perspective of general pathology and immunopathology. The consideration of the action mechanisms of vaccines, which is connected to the plausibility that a response to stress can be excessive or distorted in some cases, suggests that some aspects of the WHO procedure of causality assessment are inadequate to deal with this complexity. The rigid exclusion criteria which occurs in some steps of the algorithm (such as the “another cause” of AEFI in step I and the negative evidence in literature) can be a source of errors, or at least questionable interpretations, especially when the clinical situation or the autopsy are not clear and decisive. Error of evaluation would be to consider as “the cause” of AEFI any pathology that may be present at the time of vaccination, without considering the possible interaction between this pathology and the effect of the vaccine as a possible contributing cause.\n\nThree case studies of causality assessment have been reported here (Box 1), which led to an exclusion of the causal link. The few data that were officially communicated by the regulatory authorities (AIFA) in their reports do not allow direct criticism of the conclusions reached, but are sufficient to illustrate the difficulties that may arise, in practical terms, in applying the algorithm in cases of complex clinical situations. Especially if the clinic or laboratory indicates that the AEFI derives from a multifactorial pathogenesis, it would not be correct to discard in this way a possible role of the vaccine in determining a serious adverse reaction, which obviously involves a series of conditions predisposing a body to damage. In fact, in the case of pre-existing or concomitant pathology, which can be considered a susceptibility factor, the vaccine could represent a contributory, triggering, or worsening condition.\n\nFrom these considerations, a first operative suggestion emerges. To avoid potential errors of interpretation, it would be appropriate that in the WHO guidelines of the causality assessment be explicitly specified that the “other causes” mentioned in step 1, should only be considered a reason for excluding the causal link, when they are “independent” of the possible vaccine biological action. In other words, to declare “inconsistent” the association with the vaccination, it should be excluded that the condition existing in the subject at the time of the damage may have interacted with the vaccine, enhancing its pathogenic potential (or vice versa the vaccine had worsened the pre-existing situation). Only in this situation, would it be correct to exclude the association between AEFI and vaccine action in phase I. In order for this prospect to materialize, the risk factors of vaccine reactions need to be better identified including additive (non-synergistic) and multiplicative (synergistic) forms, similarly to what has been done in other fields. Improved understanding of risk factors would contribute to reducing the uncertainties of vaccination choices, which are often perceived by the population as “leaps in the dark”.\n\nA second topic of discussion concerns the claims for compensation for vaccination damage. Due to the inherent complexity of the pathogenesis of vaccination reactions, an absolute certainty of the causal role of the vaccine is always difficult, but often it is also difficult to exclude it. Using the WHO algorithm slavishly, it is likely that many adverse events, due to various concomitant factors, will end up as “indeterminate”. This conclusion could gather many cases in which plausibly the vaccine damage may have occurred, but there is neither absolute certainty, nor adequate representation in the literature. For claimants, an “indeterminate” causal link is equivalent in practice to the conclusion of “unrelated” and is therefore potentially a reason for discrimination. The latter problem, with human and economic sides, could be addressed, for example, by considering the possibility of assigning compensation - perhaps in part - even if the hypothesis of vaccine damage is only “probable” and in any case its contributory role cannot be excluded.\n\nThe difficulties that courts encounter when deciding on compensation claims in which scientific uncertainty is present are noteworthy, also because the case law of different countries like Germany and France diverge with regard to their relationship to scientific criteria of causality4. In the Italian system, in the matter of civil liability, it is sufficient that the causal link between fact and harmful event occurred with a probability of 50% + 1 so that Civil Liability can be affirmed (See ex multis. Cass. Civ. Sent. N. 21619/2007). The aforementioned criteria have also applied to damages, deriving from compulsory (or strongly recommended) vaccinations. Indeed, the jurisprudence of the Supreme Court of Cassation has ratified the principle with multiple judgments (see Ex multis Cass. Civ. Sec. VI Judgment no. 25119/2017; Cass. Civ. Sez. Lavoro, Judgment no. 22078/2018) according to which the existence of the causal link between the vaccination administration and the occurrence of the damage to health must be evaluated according to a criterion of reasonable scientific probability inspired by the “more likely than not” principle. It is worth noting that in 2017 a judgement of the European Court of Justice in Luxembourg allowed courts to decide that a vaccine had caused harm, taking into account the “serious, specific, and consistent” presumptions of a causal relationship, even when there is no certain proof based on medical research to support this178. The presumptions include the time frame between vaccination and the evidence of disease, a lack of family history of the disease, and a considerable number of instances of the disease appearing, after administration of the vaccine.\n\nA third series of considerations concerns legislation in which a vaccination obligation is being imposed. Clearly, this imposition implies a small but not absent risk of adverse events. In certain high-risk groups, such as immunocompromised patients and those with a history of previous anaphylactic reaction to a vaccine or its components, selective withholding of immunizations must be considered to decrease potential adverse events. However, aside from the case of primary immunodeficiency and some rare metabolic disease, till now there has been no routine laboratory test available with sufficient predictivity power to detect an increased risk of adverse reactions at the individual level. In this situation, a cautionary criterion should be adopted for all cases in which the existence of susceptibility factors is suspected, such as for example: a) family pathobiographic history, i.e. the previous occurrence of serious adverse reactions in family members, even without managing to make a molecular diagnosis, or b) genetic variation in a precise sense, determined by some already known specific polymorphism (see Table 1). It seems reasonable that, if a child presents with an increased risk of adverse reactions compared to the average risk of the population, for this subject the vaccination obligation should be “loose” and the choice of whether to vaccinate (taking the risk of AEFI) or not to vaccinate (taking the risk of any illness to which the subjects are unprotected) should be left to the doctor, in agreement with the parents. A corollary of this problem indicates that in order to improve risk assessment, studies on genetic predispositions to vaccine damage should be increased by establishing systematic analysis programs for polymorphisms, to be carried out from birth. The more subjects that are entered in these databases, after years of accumulating cases and comparing healthy and damaged subjects, the more precise the calculation of the relative risk associated with vaccinations will be. As knowledge of vaccinomics and adversomics increases, this estimate will be an increasingly precise element in decision-making in the coming years.\n\nAdverse reactions associated with the vaccine and immunization-related error events can affect healthy individuals and should be promptly identified to decide whether and how to compensate those affected. We must reiterate the need not to confuse the epidemiological and individual perspectives: one being the risk/benefit of vaccination, another is the recognition (and possible compensation) of a causal-link association between an adverse event and the vaccine. The fact that a child carries a heightened risk of vaccination, does not mean that he/she should not be vaccinated. The risk/benefit ratio must be weighed on an individual level with care and precision, especially considering the incidence and severity of diseases to which they would be exposed if unvaccinated. On the other hand, it is correct that damaged individuals - perhaps even those who just “probably” have suffered serious harm from vaccination – are recognized and compensated. This way of proceeding should also serve to increase the general confidence of the population in vaccinations and reduce litigation in the health care system.\n\n\nData availability\n\nNo data are associated with this article.",
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PubMed Abstract | Publisher Full Text\n\nHaralambieva IH, Ovsyannikova IG, Kennedy RB, et al.: Genome-wide associations of CD46 and IFI44L genetic variants with neutralizing antibody response to measles vaccine. Hum Genet. 2017; 136(4): 421–435. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScheffer IE: Diagnosis and long-term course of Dravet syndrome. Eur J Paediatr Neurol. 2012; 16(Suppl 1): S5–S8. PubMed Abstract | Publisher Full Text\n\nAuvin S, Jeljeli M, Desnous B, et al.: Altered vaccine-induced immunity in children with Dravet syndrome. Epilepsia. 2018; 59(4): e45–e50. PubMed Abstract | Publisher Full Text\n\nVerbeek NE, Jansen FE, Vermeer-de Bondt PE, et al.: Etiologies for seizures around the time of vaccination. Pediatrics. 2014; 134(4): 658–666. PubMed Abstract | Publisher Full Text\n\nAzzari C, Gambineri E, Resti M, et al.: Safety and immunogenicity of measles-mumps-rubella vaccine in children with congenital immunodeficiency (DiGeorge syndrome). 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaulatzai MA: Chronic functional bowel syndrome enhances gut-brain axis dysfunction, neuroinflammation, cognitive impairment, and vulnerability to dementia. Neurochem Res. 2014; 39(4): 624–644. PubMed Abstract | Publisher Full Text\n\nBrown GC: The endotoxin hypothesis of neurodegeneration. J Neuroinflammation. 2019; 16(1): 180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGershwin LJ: Adverse Reactions to Vaccination: From Anaphylaxis to Autoimmunity.. Vet Clin North Am Small Anim Pract. 2018; 48(2): 279–290. PubMed Abstract | Publisher Full Text\n\nSegal Y, Shoenfeld Y: Vaccine-induced autoimmunity: the role of molecular mimicry and immune crossreaction. Cell Mol Immunol. 2018; 15(6): 586–594. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee SJ, Lee S: Toll-like receptors and inflammation in the CNS. Curr Drug Targets Inflamm Allergy. 2002; 1(2): 181–191. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nGholizadeh P, Mahallei M, Pormohammad A, et al.: Microbial balance in the intestinal microbiota and its association with diabetes, obesity and allergic disease. Microb Pathog. 2019; 127: 48–55. PubMed Abstract | Publisher Full Text\n\nTozzi AE, Asturias EJ, Balakrishnan MR, et al.: Assessment of causality of individual adverse events following immunization (AEFI): a WHO tool for global use. Vaccine. 2013; 31(44): 5041–5046. PubMed Abstract | Publisher Full Text\n\nCosmacini G: Storia della medicina e della sanità in Italia. Bari, Laterza. 2010. Reference Source\n\nTourbah A, Gout O, Liblau R, et al.: Encephalitis after hepatitis B vaccination: recurrent disseminated encephalitis or MS? Neurology. 1999; 53(2): 396–401. PubMed Abstract | Publisher Full Text\n\nYuan JL, Wang SK, Guo XJ, et al.: Acute Disseminated Encephalomyelitis following Vaccination against Hepatitis B in a Child: A Case Report and Literature Review. 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PubMed Abstract\n\nShaw CA, Tomljenovic L: Aluminum in the central nervous system (CNS): toxicity in humans and animals, vaccine adjuvants, and autoimmunity. Immunol Res. 2013; 56(2–3): 304–316. PubMed Abstract | Publisher Full Text\n\nMostafa GA, Shehab AA, Al-Ayadhi LY: The link between some alleles on human leukocyte antigen system and autism in children. J Neuroimmunol. 2013; 255(1–2): 70–74. PubMed Abstract | Publisher Full Text\n\nEdmiston E, Ashwood P, Van de Water J: Autoimmunity, Autoantibodies, and Autism Spectrum Disorder. Biol Psychiatry. 2017; 81(5): 383–390. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHughes HK, Mills KE, Rose D, et al.: Immune Dysfunction and Autoimmunity as Pathological Mechanisms in Autism Spectrum Disorders. Front Cell Neurosci. 2018; 12: 405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGherardi RK, Crepeaux G, Authier FJ: Myalgia and chronic fatigue syndrome following immunization: macrophagic myofasciitis and animal studies support linkage to aluminum adjuvant persistency and diffusion in the immune system. Autoimmun Rev. 2019; 18(7): 691–705. PubMed Abstract | Publisher Full Text\n\nAoun Sebaiti SM, Abrivard M, Blanc-Durand P, et al.: Macrophagic myofasciitis-associated dysfunctioning: An update of neuropsychological and neuroimaging features. Best Pract Res Clin Rheumatol. 2018; 32(5): 640–650. PubMed Abstract | Publisher Full Text\n\nSoares Santos D, Santos A, Rebelo O, et al.: Macrophagic myofasciitis: a challenging diagnosis. BMJ Case Rep. 2018; 2018: pii. bcr-2018-224602. PubMed Abstract | Publisher Full Text\n\nAoun Sebaiti M, Kauv P, Charles-Nelson A, et al.: Cognitive dysfunction associated with aluminum hydroxide-induced macrophagic myofasciitis: A reappraisal of neuropsychological profile. J Inorg Biochem. 2018; 181: 132–138. PubMed Abstract | Publisher Full Text\n\nZinka B, Rauch E, Buettner A, et al.: Unexplained cases of sudden infant death shortly after hexavalent vaccination. Vaccine. 2006; 24(31–32): 5779–5780. PubMed Abstract | Publisher Full Text\n\nBiru N, Shlobin OA, Aryal S, et al.: Rare Case of Rapidly Progressive Interstitial Lung Disease Following Adult Tetanus, Diptheria, and Pertussis (TDAP) Vaccination. Am J Resp Crit Care Med. 2018; 197: A6582. Reference Source\n\nHibino M, Kondo T: Interstitial Pneumonia Associated with the Influenza Vaccine: A Report of Two Cases. Intern Med. 2017; 56(2): 197–201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOsawa M, Nagao R, Kakimoto Y, et al.: Sudden Infant Death After Vaccination: Survey of Forensic Autopsy Files. Am J Forensic Med Pathol. 2019; 40(3): 232–237. PubMed Abstract | Publisher Full Text\n\nPuliyel J, Phadke A: Deaths following pentavalent vaccine and the revised AEFI classification. 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}
|
[
{
"id": "61078",
"date": "18 Mar 2020",
"name": "David Legge",
"expertise": [
"Reviewer Expertise I am a generalist public health physician with a particular interest in global health policy. I have a long standing interest in complexity theory as applied to biological and social systems. However",
"I have approached this review with a policy perspective."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 suggests that the 2018 WHO algorithm for causality assessment in cases of adverse events following immunisation (AEFI) may be overly mechanistic and may not do justice to the complexity of biological systems and the possibility of multifactorial causation.\n\nHe sets the scene regarding bio-complexity with reference to complex disease causation, the complexity of the immune response (including the genesis of autoimmunity), the role of genetic variants in modulating immune reactions, and the role of the microbiome.\n\nHe then examines the 2018 WHO algorithm considering four steps in some detail.\n\nThe first step in WHO's causal algorithm asks if there is 'strong' evidence for an alternative cause of the event. The author suggests that this either-or question precludes consideration of the possibility of an interaction between the alternative cause and the immunisation event.\n\nThe second question raised concerns the significance of temporal association (between immunisation and adverse event) in determining causality. The author argues, drawing particularly on immunopathology, that some pathways of causation could operate over weeks and months rather than hours and days.\n\nThe third element of the algorithm which the author questions concerns the exclusion (with respect to causality) of cases where there is 'strong' evidence against a causal relation. The concern raised here concerns the meaning of 'strong' evidence in relation to the methodological limitations of such research.\n\nThe fourth element of the algorithm in question concerns the final classification of 'consistent with' or 'not consistent with' causality. Here the author returns to his concerns about the either-or logic upon which the algorithm rests and the need to properly accommodate multifactorial causation.\n\nIn concluding his review the author proposes further development of the WHO algorithm to better accommodate multifactorial causation. He also comments on the links between AEFI causality assessment and access to compensation for people who have experienced harm after immunisation. He suggests that the appropriate standards for causal inference for compensation purposes might not be same as those for regulatory purposes. Finally the author returns to multifactorial causation, highlighting in particular, genetic influences and the need for further research including regulatory monitoring of genetic factors (and perhaps microbiomic factors). He suggests that closer attention to the interplay of these (potentially) contributory factors could contribute to more evidence-informed decision-making regarding exemptions from mandated (or highly recommended) immunisations.\n\nThis is a very thoughtful and well informed contribution to the continuing discussion of causal assessment in cases of AEFI. The author has presented a 'strong' case for closer attention to complexity and multifactorial causation in causality assessment of AEFIs. The distinctions he makes between the different contexts and purposes of causal assessment (regulation, compensation and exemption) are useful.\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": "5363",
"date": "14 Apr 2020",
"name": "Paolo Bellavite",
"role": "Author Response",
"response": "I warmly thank Doctor Legge for this positive comment on my work, of which he demonstrates that he has understood the fundamental meaning in the context of the complex vision of multifactorial diseases. His contribution is invaluable in preparing the second version and I will certainly take it into account Paolo Bellavite (Author)"
}
]
},
{
"id": "61073",
"date": "23 Mar 2020",
"name": "Jacob Puliyel",
"expertise": [
"Reviewer Expertise Pediatrics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPaola Bellavite (Verona University, Italy) has reviewed the WHO’s causality classification of adverse events following immunization (AEFI) in an opinion piece. Using 3 case studies of AEFI deaths reported to the Italian Medicines Agency (Agenzia Italiana del Farmaco AIFA) he illustrates how application of the WHO algorithm is difficult and prone to error.\n\nThe glossary of the WHO manual (page vii) defines ‘causal association’ as a cause and effect relationship between causative factor and a disease with no other factor intervening in the process. Bellavite, quite rightly, feels that this is a wrong approach.\n\nHe lists (Table 1), a series of genetic disorders that have been associated with tendency to develop AEFI. Using the WHO definition, a causal association with vaccine would be denied because of the genetic factor intervening in the process. Bellavite has proposed that a ‘consistent association of the adverse event with the vaccine’ must only be excluded when the presumed ‘other cause’ independently (without interaction with the vaccine) causes the AEFI. This makes good sense.\n\nAt step 3 along the mandatory path of the algorithm, the question is: “Is there strong evidence against a causal association?” Bellavite correctly points out the impossibility of proving a negative. “Lack of evidence of association” may mistakenly be considered as “evidence of lack of association” or evidence against a causal association.\n\nIt is interesting that the WHO manual quotes an Institute of Medicine (IOM) report on a study looking at a possible relationship between SIDS and vaccines and which concluded that vaccines did not cause SIDS. Will this loose use of the generic term ‘vaccines’ mean that, hereinafter, no vaccine can have a causal association with SIDS or does this statement relate only to the vaccines examined by the IOM. The problem with proving a universal negative is that a single instance of a positive association can negate all the previous experiences and studies. Such a universal negative assertion is seldom made in scientific literature.\n\nThese are important issues that have been raised.\n\nTill 2013 the WHO used the Brighton classification of AEFI (1) and causal association was classified as ‘certain’: ‘probable’: ‘possible’: ‘unlikely’ and ‘unclassifiable’. The categories were revised in 2013 (2). F1000research published a critique of this classification by the reviewer (3). The Second Edition of Revised AEFI classification was published (with minor changes) in 2018 (4).\n\nIn a communication in the British Medical Journal, Chandler of the Brighton Collaboration has asserted (5) and I quote extensively (italicized):\n\n“The WHO AEFI causality assessment was developed by the Vaccines Safety Group at the WHO with the support of the Global Advisory Committee on Vaccine Safety. The target user group for this classification system are persons working in countries in whom vaccines are administered via WHO sponsored public health programmes. Those persons are largely concerned with the detection of \"signals\" of changes in frequency of the more common, expected events which could suggest vaccine quality-related problems, immunisation errors, or multi-use vial contamination, etc. At the current time, most AEFI reports collected and assessed with the WHO AEFI Causality Classification remain within the databases of the public health programmes and are not forwarded into the databases of the national pharmacovigilance centres of most lower and middle income countries. In contrast, more general guidance for causality assessment, such as the WHO-UMC causality criteria and the Naranjo algorithm, were developed by various groups working within the greater field of pharmacovigilance. The target user groups for these classification systems are those persons working within national pharmacovigilance centres, usually working within or collaboratively with national regulatory centres, and responsible for post-marketing safety surveillance of both drugs and vaccines used within their countries. Within such centres adverse event reports for drugs and vaccines are often maintained within a single database (one notable exception being the USA), and causality assessment is approached in a similar way for all products. Detection of \"signals\" within the database can be conducted qualitatively (on a \"case-by-case\" basis) and/or quantitatively (via statistical screening ). Higher income countries which do not rely upon implementation of vaccine administration through WHO public health programmes will handle reports of AEFI through these national pharmacovigilance centres. Furthermore, it is worth noting that most reports of AEFI contained with Vigibase, the database of individual case safety reports for the WHO Programme of International Drug Monitoring, are from countries who channel reports of AEFI through their national pharmacovigilance system, and therefore most reports within the global database have not been subject to WHO AEFI causality assessment. Taking the specific example of narcolepsy, reports of this condition in association with Pandemrix, an H1N1 pandemic vaccine, were initially received into the national pharmacovigilance centres of Sweden and Finland, and therefore they were not subject to causality assessment by the WHO AEFI classification system. This signal was detected, in fact, because these clusters of reports in young children were \"unexpected\" , by both the reporting physicians (based upon their clinical practice) and by the regulators (based upon the expected reporting patterns within their national databases of suspected adverse drug reactions). The current system referred to as \"robust\" within this analysis therefore refers to practice of vaccine pharmacovigilance by national pharmacovigilance/regulatory centres, not that of national immunisation centres routinely utilising the WHO-AEFI causality classification system.”\n\nIt seems from this that the WHO causality assessment is meant for poor and developing countries and most reports within the global database for pharmacovigilance have not been subject to WHO AEFI causality assessment. it is interesting that the cases cited by Bellavite, the AEFI deaths reported to the Italian Medicines Agency were subjected to the WHO AEFI assessment.\nThe point that Bellavite makes is that compensation may be denied to families who die after vaccination, utilizing this classification. It will be intriguing to know if this classification is used in Italy to deny compensation but, as a ‘developed country’, it uses a second system for pharmacovigilance.\n\nThe 2018 revised manual says it was ‘scientifically evaluated’ looking for inter-rater reliability between teams from India and Zimbabwe. It was not examined against any gold standard. If two populations consistently perceive the world is flat, it does little validate the ‘scientific’ reliability of that perception.\n\nThe paper by Bellavite is an important addition to the literature. However, it can be improved by extensive revision. The language can be improved and corrected in many places. This reviewer has often had to resort to such help, for his scientific communications.\n\n1. Introduction: The author writes that AEFI harms a few “unlucky” individuals. The term related to luck put within quotation marks is best deleted in a scientific communication.\n\n2. Page 4 Innate immune response. It is not clear what the author wants to convey about the risk of fever after MMR. He says this is more in children under 35 months compared to children older than 4 years of age. I am not able to understand what this has to do with the AEFI classification and why this is brought up here.\n\n3. Page 5 The author suggests that some autoimmune disorders may be associated with immunization but it is not specified what changes in the AEFI classification will help to identify the role that vaccines play.\n\n4. The list of genetic disorders listed in Table 1 is useful as a ready-reckoner, but for that, it must be as exhaustive as possible. I am not an expert in this area but the association of AEFI with mitochondrial disorders is one that I recognize is missing from the list (Poling PMID 16566887)\n\n5. Page 7 The text says “It is important to point out vaccines may safely be administered in children with Di George syndrome.” Why is it important to state this? There are a whole host of genetic disorders where vaccines can be administered with impunity. Why has Di Gorge been singled out to be declared as safe\n\n6. Micrbiome - The relevance of the paragraph on the microbiome is also not clear in the context of AEFI classification.\n\n7. Page 8 The first two paragraphs: It is not clear what the author wants to convey and how it relates to the WHO AEFI classification method\n8. Page 9 Top paragraph not clear\n\n9. So also Note 3 The Literature (Delete ‘Note 3’ from the paragraph heading)\n\nThe content of this paragraph is not clear\n\n10. Page 10 Second last paragraph The author writes\n\n“The most obvious case of a possible overlap between autism spectrum symptoms and another disease, surely caused by vaccine adjuvants, is the macrophagic myofasciitis.“\nA little more elaboration would be helpful because macrophagic myofasciitis is a relatively new syndrome associated with vaccine aluminium adjutants and its association with cognitive disorders is known even less.\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? Partly",
"responses": [
{
"c_id": "5385",
"date": "14 Apr 2020",
"name": "Paolo Bellavite",
"role": "Author Response",
"response": "I thank Dr. Puliyel for his reviewer’s report that allows me to improve the paper and specify some aspects better. Here I respond to his observations point-by-point and highlight the new parts inserted in the text I appreciate that the most important proposal of the paper has been accepted. Rightly dr. Pulijel recalls that the same F1000research Journal published a critique of this classification, which I appropriately cited (quotation 158 of the first version). There are several points raised by the Reviewer that I have addressed in the revised paper: 1. Dr. Puliyel has cited a long and interesting declaration of dr. Chandler including the sentence “The target user group for this classification system are persons working in countries in whom vaccines are administered via WHO sponsored public health programmes “ and suggested that “ It seems from this that the WHO causality assessment is meant for poor and developing countries and most reports within the global database for pharmacovigilance have not been subject to WHO AEFI causality assessment. it is interesting that the cases cited by Bellavite, the AEFI deaths reported to the Italian Medicines Agency were subjected to the WHO AEFI assessment.” Response: This is a straightforward point that was raised also by dr. Chandler as Reviewer and I have modified the text according your and her suggestions. I have given only one response, under her comments to the paper. Moreover, connected with Chandler’s comments, I have added to the Discussion a paragraph to better clarify the limitations of WHO algorithm for evaluating the responsibility of vaccines in multifactorial AEFI: “The WHO causality assessment is mainly constructed on a concept of direct ‘a cause-and-effect relationship”, thus dismissing the multifactorial nature of inflammatory and immune phenomena. By underestimating interacting causes, the method classifies a possible association as “inconsistent” when there is another cause and as “indeterminate” when the role of the vaccine can’t be excluded, but there is no proof that it is “the” cause. Using this definition of causal association, many adverse events, where the vaccine plays a role as con-cause, remain unrecognized. Others (Puliyel and Naik, 2018) have noted that, according to this scheme, an acute cardiac decompensation after influenza vaccination in an elderly person with chronic cardiac failure might not be considered as causally related to the vaccine. Similarly, sudden death after vaccination of an infant with pre-existing heart disease might not have relationship with the vaccine. Furthermore, the contribution of vaccine in precipitating encephalopathy in patients who are susceptible on account of genetic factors will also not be considered. If this type of problem occurs, in addition to causing detriment to a injured person, it leads to an overall underestimation of the risks of a given vaccine. 2. The point that Bellavite makes is that compensation may be denied to families who die after vaccination, utilizing this classification. It will be intriguing to know if this classification is used in Italy to deny compensation but, as a ‘developed country’, it uses a second system for pharmacovigilance. Response: In Italy, the WHO classification is considered the “gold standard” in the evaluation of AEFI originating from pharmacovigilance reports, so it is normally the only one that is used for causality assessment. Of course, if people who believe they have suffered unrecognized damage from vaccination appeal to a court of justice, a much more detailed assessment follows, where the expert consultants of the parties are challenged with all the available documentation. I have added this further explanation to the Discussion in the paragraphs concerning the compensation. 3. The 2018 revised manual says it was ‘scientifically evaluated’ looking for inter-rater reliability between teams from India and Zimbabwe. It was not examined against any gold standard. If two populations consistently perceive the world is flat, it does little validate the ‘scientific’ reliability of that perception. Response: I agree that this is one of the several problems raised by the manual and I hope that my paper will contribute to highlight that considering the WHO classification as an universal “gold standard” would be an error. 4. Introduction: The author writes that AEFI harms a few “unlucky” individuals. The term related to luck put within quotation marks is best deleted in a scientific communication Response: OK I agree and have removed this word 5. Page 4 Innate immune response. It is not clear what the author wants to convey about the risk of fever after MMR. He says this is more in children under 35 months compared to children older than 4 years of age. I am not able to understand what this has to do with the AEFI classification and why this is brought up here. Response: I wanted to report in one sentence the quantitative value of the risk of febrile seizures according to the literature and in the following one the risk of MMRV compared to MMR alone. I believe it is important to offer readers an estimate of what the frequency of one of the most serious adverse reactions is, because the evaluation of causality must also take into account, among the other factors, also the probability that a certain AEFI can occur in a specific age group. 6. Page 5 The author suggests that some autoimmune disorders may be associated with immunization but it is not specified what changes in the AEFI classification will help to identify the role that vaccines play. Response: This topic is very important but does not have an easy solution. In fact, as has been reported in the text, autoimmune diseases and in general chronic post-vaccination chronic syndromes are due to multiple factors intertwined with each other and, moreover, they can arise weeks or months after vaccination. However, to answer the reviewer’s correct question, I have added a paragraph on autoimmune diseases to the \"final categorization\" in Discussion (Note 4.) and on page 5 I have added a sentence that refers to the final discussion: “The implications of these concepts in the causality assessment are discussed in a later chapter (see Note 4.)” The added paragraph is the following: “As mentioned, multifactorial diseases, such as autoimmune diseases, are often conditioned by various genetic and acquired factors. In these cases, the role of vaccination could be to slatentize a predisposing condition, which would have led to the disease slower or would not even appear. If this is the case, it is probable that the case study will neither confirm nor deny the role of the vaccine, so that the causality assessment would come to the conclusion of an \"undetermined\" association. Obviously, this procedure, if applied systematically to a series of cases, would lead to an underestimation of the etiological role of vaccines in autoimmune diseases. To overcome this vicious circle, in the final categorization (phase IV), the probability that the vaccine played a role in determining the event could be assessed and scored, taking into account the other possible factors involved. In this way, it would prevent information on the partial role of the vaccine, obtained from a particular case, from being lost in the study of a series of cases.” 7. The list of genetic disorders listed in Table 1 is useful as a ready-reckoner, but for that, it must be as exhaustive as possible. I am not an expert in this area but the association of AEFI with mitochondrial disorders is one that I recognize is missing from the list (Poling et al., 2006) Response: I thank of this suggestion, I have added the indicated reference to Table 1. To the best of my knowledge, there are not further genetic traits which have been associated with AEFI. 8. Page 7 The text says “It is important to point out vaccines may safely be administered in children with Di George syndrome.” Why is it important to state this? There are a whole host of genetic disorders where vaccines can be administered with impunity. Why has Di Gorge been singled out to be declared as safe Response. Also this question is correct. The reason is that this is a genetic disorder of immune system (immunodeficiency), it is a notable exception of the rule that live vaccinas should not be administered to these children. In any case, to better clarify this point, in the revised version I have changed “in this context” (too generic) to “in the context of immunodeficiency diseases” 9. Microbiome - The relevance of the paragraph on the microbiome is also not clear in the context of AEFI classification. Response: Indeed, this point needs to be clarified. Thanks to the reviewer's question, I added this explanation at the end of the microbiome paragraph: “The role of microbiome is important from the perspective of susceptibility factors of AEFI, because it is possible that an alteration of the gut health, especially with the leak of endotoxins in the general circulation, increases the susceptibility to a stronger and more serious reaction to the immune stimulus represented by the vaccine . Under these predisposing conditions the plausibility that a serious inflammatory reaction may be triggered by a vaccination increases.” 10. Page 8 The first two paragraphs: It is not clear what the author wants to convey and how it relates to the WHO AEFI classification method R: This topic is central and has to do precisely with the complex nature of many reactions to vaccines. Also thanks to the review to the first version by Dr. David Legge, who appreciated the setting of the entire work based on the description of the typical features of complex systems, I believe that this difficult topic has been well developed. Since it is possible that the meaning of this point may still be a little obscure, I added an explanatory paragraph, which is more closely related to the AEFI causal assessment: “Therefore, in the case of diseases involving several possible causes and/or mechanisms, the purpose of AEFI classification cannot be to identify \"the\" determining cause, but it may be more correct to try to establish with what probability one or more the factors involved (vaccines, genetic or epigenetic traits, previous or concomitant infections, drugs, age, gender, nutrition and metabolism, etc.) may have contributed to the occurrence of the event. “ 11. 8. Page 9 Top paragraph not clear Response. I have added a further paragraph to better clarify the problem of time window: “The WHO guidelines consider this problem under the question \"In this patient, did the event occur within a plausible time window after vaccine administration?\" and in a note cite as a \"detailed document\" a book of the Institute of Medicine(Institute of Medicine, 2012). However, in that document there are no indications on the suitable time windows of autoimmune diseases or in general of chronic diseases following vaccinations. In some cases, causality is excluded by using rather short time windows. For example, the case of a man with symptoms of chronic inflammatory disseminated polyneuropathy that occurred 8 weeks after a tetanus toxoid vaccine(Hughes et al., 1996) is presented and it is argued that this interval is \"too long\". However, autoimmune diseases and chronic post-vaccination syndromes in general can occur several weeks or months after vaccination. In cases of fibromyalgia and chronic fatigue disease following hepatitis B vaccination (Agmon-Levin et al., 2014), the time interval between vaccination and the onset of symptoms was 38.6 days, but with a large time interval (+/- 79.4 days). In a systematic prospective case-referent study conducted to assess the risks of autoimmunity associated with HPV vaccines, a reasonable time window of 24 months for multiple sclerosis, connective tissue disease, type-1 diabetes, and thyroiditis was adopted (Grimaldi-Bensouda et al., 2017). This paper excluded an association between HPV vaccination and these disease, but an increased percentage of cases had personal or family (in first-degree relatives) history of autoimmunity (14.7% of cases versus 7.2 % of referent group, p <0.05), endorsing the importance of genetic susceptibility to vaccine adverse effects. Given the complexity and multifactoriality of chronic autoimmune diseases and the lack of precise references on the time frame of appearance of these diseases after vaccination, the possibility could be considered that, in an upcoming edition of the guidelines, it is specified that the time window for autoimmune diseases should be sufficiently large (e.g. 24 months(Grimaldi-Bensouda et al., 2017) ) to not exclude slow-onset cases, or that a restricted time frame should be applied only to AEFI with acute onset (for example: hyperthermia, febrile seizures, anaphylaxis). 12. So also Note 3 The Literature (Delete ‘Note 3’ from the paragraph heading) The content of this paragraph is not clear Response: To clarify the meaning of the paragraph, at the beginning I have added a sentence: ” In practice, if there is published evidence in literature that rejects a statistically significant association between a disease and previous vaccinations, this argument could be used to exclude in each particular case that vaccination may have caused the reported disease. The evidence at population scale is used at individual level.” I cannot delete the words \"Note 3\" because the heading helps to clarify that this paragraph connects with the third step of the algorithm and the whole chapter is divided into four notes in their order. 13. Page 10 Second last paragraph. The author writes “The most obvious case of a possible overlap between autism spectrum symptoms and another disease, surely caused by vaccine adjuvants, is the macrophagic myofasciitis.“ A little more elaboration would be helpful because macrophagic myofasciitis is a relatively new syndrome associated with vaccine aluminium adjuvants and its association with cognitive disorders is known even less. Response: This topic was developed in a previous chapter, where the pathogenic effects of aluminum on the central nervous system were described, including cognitive dysfunction, sensory disturbances, and motor retardation. I added this sentence to the discussion at that point mentioned by the Reviewer. I believe these changes help make the text clearer and I thank the Reviewer for that Reference List Agmon-Levin N, Zafrir Y, Kivity S, Balofsky A, Amital H, Shoenfeld Y. Chronic fatigue syndrome and fibromyalgia following immunization with the hepatitis B vaccine: another angle of the 'autoimmune (auto-inflammatory) syndrome induced by adjuvants' (ASIA). Immunol Res 2014; 60(2-3): 376-383. Grimaldi-Bensouda L, Rossignol M, Kone-Paut I, Krivitzky A, Lebrun-Frenay C, Clet J, Brassat D, Papeix C, Nicolino M, Benhamou P Y, Fain O, Costedoat-Chalumeau N, Courcoux M F, Viallard J F, Godeau B, Papo T, Vermersch P, Bourgault-Villada I, Breart G, Abenhaim L. Risk of autoimmune diseases and human papilloma virus (HPV) vaccines: Six years of case-referent surveillance. J Autoimmun 2017; 79: 84-90. Hughes R A, Choudhary P P, Osborn M, Rees J H, Sanders E A. Immunization and risk of relapse of Guillain-Barre syndrome or chronic inflammatory demyelinating polyradiculoneuropathy. Muscle Nerve 1996; 19(9): 1230-1231. Institute of Medicine. Adverse effects of vaccines: Evidence and causality. The National Academies Press, Washington, DC 2012. Poling J S, Frye R E, Shoffner J, Zimmerman A W. Developmental regression and mitochondrial dysfunction in a child with autism. J Child Neurol 2006; 21(2): 170-172. Puliyel J, Naik P. Revised World Health Organization (WHO)'s causality assessment of adverse events following immunization-a critique. F1000Res 2018; 7: 243."
}
]
},
{
"id": "61079",
"date": "23 Mar 2020",
"name": "Christopher Exley",
"expertise": [
"Reviewer Expertise I work on human exposure to aluminium and this includes an active research programme on the safety of aluminium adjuvants used in vaccinations."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is one significant omission from this manuscript and it concerns the availability of quality peer reviewed science in the field of AEFI.\n\nHow do we know if a vaccine is the origin of AEFI if the vaccine has not been subject to clinical trials involving true placebos. The vehicle for most vaccines, probably all injected vaccines, is 0.9% NaCl. How many vaccine safety trials have used 0.9% NaCl as the placebo? The injection of 0.5mL of 0.9% NaCl IM or SC should be close to being 100% safe. The needle may damage a capillary and this could produce mild inflammation at the injection site. This could be uncomfortable for a short period of time, there may be some individuals where their genetic make-up means that the discomfort might last a little longer. However, for the vast majority of recipients, 99.9%, the injection of 0.5mL of 0.9% NaCl is absolutely safe. This is the gold standard placebo against which the safety of all vaccines should be tested and ensured. Of course, the reality is totally different. In my field of interest, vaccines that include an aluminium salt as an adjuvant, there have not been any clinical trials where a vaccine has been tested against a true placebo. I say this with the proviso that in one trial of the HPV vaccine Gardasil there is a report, filed with the Clinical Trials Database, where a small number of trial participants did receive a saline placebo. These individuals reported no (zero) AEFI, while the vaccine group and the Al adjuvant only group reported an incidence of ca 2.5% AEFI.\n\nThe reality is that the design of causality assessment of AEFI is purposely flawed to give both the impression that all vaccines are safe and, of course, to allow those conducting the trials (who are never independent) to decide which of the many and serious AEFI occurring regularly might be attributed to the vaccine. Vaccines are inherently unsafe and rather than investing in improving their safety the industry has devised a mechanism to disguise their toxicity. I do wonder why the author has not included some discussion of this in his otherwise well informed text.\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? Partly",
"responses": [
{
"c_id": "5386",
"date": "14 Apr 2020",
"name": "Paolo Bellavite",
"role": "Author Response",
"response": "I acknowledge dr. Christopher Exley for his comment, asserting that there is one significant omission from the first edition of the paper, concerning the availability of quality peer reviewed science in the field of AEFI. I agree with his concerns, so I have included a specific paragraph at the end of the Note 3, devoted to the role of medical literature in the overall procedure : “The WHO manual of causality assessment refers to the peer reviewed literature to evaluate whether there is evidence of association between vaccine and pathology (step 2 of the algorithm) or if there is opposing evidence (step 3). However, this utilization of supposed “evidence” may be flawed, since the safety of vaccines is normally proven with clinical trials that are not conducted by comparisons with a true placebo, such as physiological solution (0.9% NaCl). The latter is the “gold standard” placebo against which the safety of all vaccines should be tested and ensured, but the reality is different, especially for those vaccines that contain adjuvants. For example, the safety of the HPV vaccine Gardasil was tested in 6 clinical trials, in 5 of which the control group received Aluminum Hydroxyphosphate Sulfate, while in only one of which the physiological solution was used as placebo. However, in the summary of the safety profile of the vaccine (available from : http://www.merck.com/product/usa/pi_circulars/g/gardasil/gardasil_pi.pdf, accessed 2020 April 2), the systemic and serious adverse effects, namely the rate of autoimmune disorders, are evaluated comparing the group receiving Gardasil with only one group, receiving aluminium or placebo. By this way, any potential reactogenic effect of aluminium salts was masked. Certainly, vaccine safety is assessed also in the post-marketing phase by means of pharmacovigilance systems, which can provide important indications on the incidence of AEFI in vaccinated subjects, which can be compared with unvaccinated subjects. However, this type of comparison is largely subject to various types of selection bias and to the diversity of non-randomized groups. The problem becomes even more serious in the case of adverse reactions with low incidence. For these methodological reasons, the application of the evidence from medical literature to assess causality should be used with great caution and should not become a cut-off argument to establish or exclude causality.” I hope thar after this requested implementation, the second version of the manuscript is considered as acceptable."
}
]
},
{
"id": "61076",
"date": "31 Mar 2020",
"name": "Rebecca Chandler",
"expertise": [
"Reviewer Expertise Pharmacovigilance",
"vaccine safety surveillance"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCausality assessment of adverse events following immunization: the problem of multifactorial pathology.\n\nIn his opinion piece the author has addressed the insufficiencies of the WHO AEFI causality algorithm to appreciate the complexities manner in which vaccines cause harm specifically those which are related to an “excessive or biased inflammatory and immune response” to the vaccine product. He writes from the perspective of a pathologist, specialists which are often involved in cases of compensation claims relating to individual level questions of causality. Of particular concern to the author, as demonstrated in the 3 case examples provided, are questions relating to the role of the vaccine as one of potentially multiple factors responsible for the clinical manifestation of an AEFI. In the words of the author: ”As far as vaccines are concerned, the fact that severe reactions affect only a few individuals suggests in most cases vaccines are not the only cause of the event and further factors are necessary in the development of pathology”. He makes a number of proposals for revisions to the causality algorithm: 1) causality assessment could exclude a consistent association of the adverse event with the vaccine only when the presumed \"other cause\" is independent of an interaction with the vaccine and 2) the scientific literature should be viewed not as an exclusion criterion but as a comprehensive analysis of all the evidence for or against the role of the vaccine in causing an adverse reaction.\nOverall, the opinion of the author is agreed. However, I believe that the manuscript would benefit from some acknowledgement or proposal for revision/clarification of the primary target users of the current WHO AEFI algorithm. The WHO AEFI causality assessment was developed by the Vaccines Safety Group at the WHO with the support of the Global Advisory Committee on Vaccine Safety. The target user group for this classification system are persons working in countries in whom vaccines are administered via WHO sponsored public health programmes (largely LMIC). Those persons are largely concerned with the detection of \"signals\" of changes in frequency of the more common, expected events which could suggest vaccine quality-related problems, immunisation errors, or multi-use vial contamination, etc. A prioritisation of the identification of such AEFI can be understandable to monitor the safety of vaccines for which there is several years of experience and safety surveillance from earlier use in HIC (such as conjugate pneumococcal vaccines, HPV vaccines). Within this framework, the burden of responsibility for surveillance for the rare and unexpected AEFI has fallen to countries (and in fact vaccine manufacturers) with greater resources.\nIn contrast, higher income countries which do not rely upon implementation of vaccine administration through WHO public health programmes will handle reports of AEFI through these national pharmacovigilance centers. As a result, more general guidance is used for causality assessment, such as the WHO-UMC causality criteria and the Naranjo algorithm which developed by various groups working within the greater field of pharmacovigilance. Within such centers adverse event reports for drugs and vaccines are often maintained within a single database (one notable exception being the USA), and causality assessment is approached in a similar way for all products. Within this arrangement, the surveillance systems are better adapted for the detection of the rare and unexpected.\nI would like the author to reflect on the manner in which AEFI reports are handled in Italy, as it would be expected that vaccine reports originating from that country would be subject to collection, analysis and assessment standards endorsed by the European Medicines Agency. With the increasing likelihood of the need to implement new vaccines directly into lower income settings, the requirement of surveillance systems in these countries to detect the “rare and unexpected” is apparent. The author could address this aspect as a way to emphasize the importance of the revisions proposed.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "5387",
"date": "14 Apr 2020",
"name": "Paolo Bellavite",
"role": "Author Response",
"response": "I am glad to see that overall, my opinion is agreed. I thank for the suggestions of the Reviewer and I have revised my paper accordingly. I added two paragraphs to the text of Discussion: The debate on the best methods of surveillance in the field of vaccinology should remain open, in the interest of the entire population. In the British Medical Journal, dr. Rebecca Chandler has asserted Chandler, 2019 that the target user group for the WHO classification system are persons working in countries in whom vaccines are administered via WHO sponsored public health programmes. Those persons are largely concerned with the detection of \"signals\" of changes in frequency of the more common, expected events which could suggest vaccine quality-related problems, immunisation errors, or multi-use vial contamination, etc. It seems from this that the WHO causality assessment is meant for poor and developing countries and most reports within the global database for pharmacovigilance have not been subject to WHO AEFI causality assessment. In contrast, higher income countries which do not rely upon implementation of vaccine administration through WHO public health programmes will handle reports of AEFI through these national pharmacovigilance centers. As a result, more general guidance is used for causality assessment, such as the Naranjo algorithm (Naranjo et al., 1981,Belhekar et al., 2014) and the WHO-UMC criteria developed by various groups working within the greater field of pharmacovigilance. The WHO-UMC scale (https://www.who.int/medicines/areas/quality_safety/safety_efficacy/WHOcausality_assessment.pdf) has been developed in consultation with the National Centres participating in the Programme for International Drug Monitoring and is meant as a practical tool for the assessment of case reports. It offers a simple methodology taking into account the clinical-pharmacological aspects of the case history and the quality of the documentation of the observation (Puliyel and Naik, 2018). Within this arrangement, other criteria such as previous knowledge and statistical chance play a less prominent role in the system, so the surveillance systems are better adapted for the detection of the rare and unexpected events. Despite there is no universally accepted method for causality grading of adverse drug reactions, the WHO algorithm is now recommended specifically for the pharmacovigilance of vaccine adverse events and is increasingly used by researchers and epidemiologists worldwide, in Lower Middle Income Countries like India (Singh et al., 2018; Sebastian et al., 2019) but even in developed countries. For example, the WHO causality assessment guidelines are widely utilized in Italy (Lombardi et al., 2019;Stefanizzi et al., 2019), Germany(Mentzer et al., 2018), Canada (MacDonald and Law, 2017), and were recommended by the Brighton Collaboration Group for analysis of safety data of vaccines in pregnancy (Jones et al., 2016). Given the importance and universal utilization of this approach and its inadequacies in the evaluation of multifactorial diseases, the WHO manual needs to be urgently reevaluated and revised. References Belhekar, M. N., S. R. Taur, and R. P. Munshi, 2014, A study of agreement between the Naranjo algorithm and WHO-UMC criteria for causality assessment of adverse drug reactions: Indian J Pharmacol, v. 46, no. 1, p. 117-120. Chandler, R. E., 2019, Modernising vaccine surveillance systems to improve detection of rare or poorly defined adverse events: BMJ, v. 365, p. l2268. Jones, C. E. et al., 2016, Guideline for collection, analysis and presentation of safety data in clinical trials of vaccines in pregnant women: Vaccine, v. 34, no. 49, p. 5998-6006. Lombardi, N. et al., 2019, Vaccines Safety in Children and in General Population: A Pharmacovigilance Study on Adverse Events Following Anti-Infective Vaccination in Italy: Front Pharmacol, v. 10. MacDonald, N. E., and B. J. Law, 2017, Le système de innocuité vaccinale canadien en huit étapes : des notions pour les travailleurs de la santé: Paediatr.Child Health, v. 22, no. 4, p. e17-e20. Mentzer, D., D. Oberle, and B. Keller-Stanislawski, 2018, Adverse events following immunisation with a meningococcal serogroup B vaccine: report from post-marketing surveillance, Germany, 2013 to 2016: Euro.Surveill, v. 23, no. 17. Naranjo, C. A., U. Busto, E. M. Sellers, P. Sandor, I. Ruiz, E. A. Roberts, E. Janecek, C. Domecq, and D. J. Greenblatt, 1981, A method for estimating the probability of adverse drug reactions: Clin.Pharmacol Ther., v. 30, no. 2, p. 239-245. Puliyel, J., and P. Naik, 2018, Revised World Health Organization (WHO)'s causality assessment of adverse events following immunization-a critique: F1000Res., v. 7, p. 243. Sebastian, J., P. Gurumurthy, M. D. Ravi, and M. Ramesh, 2019, Active surveillance of adverse events following immunization (AEFI): a prospective 3-year vaccine safety study: Ther.Adv.Vaccines Immunother., v. 7. Singh, A. K., A. L. Wagner, J. Joshi, B. F. Carlson, S. Aneja, and M. L. Boulton, 2018, Causality assessment of serious and severe adverse events following immunization in India: a 4-year practical experience: Expert.Rev.Vaccines, v. 17, no. 6, p. 555-562. Stefanizzi, P., P. Stella, D. Ancona, K. N. Malcangi, F. P. Bianchi, N. S. De, D. Ferorelli, C. A. Germinario, and S. Tafuri, 2019, Adverse Events Following Measles-Mumps-Rubella-Varicella Vaccination and the Case of Seizures: A Post Marketing Active Surveillance in Puglia Italian Region, 2017-2018: Vaccines (Basel), v. 7, no. 4."
}
]
}
] | 1
|
https://f1000research.com/articles/9-170
|
https://f1000research.com/articles/9-145/v1
|
25 Feb 20
|
{
"type": "Research Article",
"title": "In silico identification of vaccine targets for 2019-nCoV",
"authors": [
"Chloe H. Lee",
"Hashem Koohy",
"Chloe H. Lee"
],
"abstract": "Background: The newly identified coronavirus known as 2019-nCoV has posed a serious global health threat. According to the latest report (18-February-2020), it has infected more than 72,000 people globally and led to deaths of more than 1,016 people in China. Methods: The 2019 novel coronavirus proteome was aligned to a curated database of viral immunogenic peptides. The immunogenicity of detected peptides and their binding potential to HLA alleles was predicted by immunogenicity predictive models and NetMHCpan 4.0. Results: We report in silico identification of a comprehensive list of immunogenic peptides that can be used as potential targets for 2019 novel coronavirus (2019-nCoV) vaccine development. First, we found 28 nCoV peptides identical to Severe acute respiratory syndrome-related coronavirus (SARS CoV) that have previously been characterized immunogenic by T cell assays. Second, we identified 48 nCoV peptides having a high degree of similarity with immunogenic peptides deposited in The Immune Epitope Database (IEDB). Lastly, we conducted a de novo search of 2019-nCoV 9-mer peptides that i) bind to common HLA alleles in Chinese and European population and ii) have T Cell Receptor (TCR) recognition potential by positional weight matrices and a recently developed immunogenicity algorithm, iPred, and identified in total 63 peptides with a high immunogenicity potential. Conclusions: Given the limited time and resources to develop vaccine and treatments for 2019-nCoV, our work provides a shortlist of candidates for experimental validation and thus can accelerate development pipeline.",
"keywords": [
"Coronavirus",
"adaptive immunity",
"immunogenicity",
"T cell cross-reactivity",
"vaccine development"
],
"content": "Introduction\n\nThe emergence and rapid spread of the recent novel coronavirus known as 2019-nCoV has posed a serious global health threat1 and has already caused a huge financial burden2. It has further challenged the scientific and industrial community for quick control practices, and equally importantly to develop effective vaccines to prevent its recurrence. In facing a rapid epidemical outbreak to a novel and unknown pathogen, a key bottleneck for a proper and deep investigation, which is fundamental for vaccine development, is the limited -- to almost no -- access of the scientific community to samples from infected subjects. As such, in silico predictions of targets for vaccines are of high importance and can serve as a guidance to medical and experimental experts for the best and timely use of the limited resources.\n\nIn this regard, we report our recent effort to computationally identify immunogenic and/or cross-reactive peptides from 2019-nCoV. We provide a detailed screen of candidate peptides based on comparison with immunogenic peptides deposited in the Immune Epitope Database and Analysis Resource (IEDB) database including those derived from Severe acute respiratory syndrome-related coronavirus (SARS CoV) along with de novo prediction from 2019-nCoV 9-mer peptides. Here, we found i) 28 SARS-derived peptides having exact matches in 2019-nCoV proteome previously characterized to be immunogenic by in vitro T cell assays, ii) 22 nCoV peptides having a high sequence similarity with immunogenic peptides but with a greater predicted immunogenicity score, and iii) 44 + 19 nCoV peptides predicted to be immunogenic by the iPred algorithm and 1G4 TCR positional weight matrices respectively.\n\n\nResults\n\nWe collected all peptides in IEDB (3, as of 13-02-2020) reported positive in T cell assays and have human as the host organism. We then conducted a local sequence alignment of 10 2019-nCoV open reading frames (ORFs) against 35,225 IEDB peptides, and found 28 exact matches. Surprisingly, all identical hits (towards target peptide length > 3) were from SARS-CoV (Table 1, Data Table 14). These peptides have been shown to bind various HLA alleles, although with higher tendency towards HLA-A:02:01, from both class I and class II, and can be target for CD8+ and CD4+ T cells respectively.\n\n*SARS-CoV: Severe acute respiratory syndrome coronavirus\n\nIn addition to 28 identical hits against SARS CoV, we observed a long tail in distribution of normalized alignment scores between 10 2019-nCoV ORFs and 35,225 IEDB peptides (Figure 1A). We therefore set out to further investigate potential vaccine targets among highly similar sequences.\n\nA. Comparison of normalized sequence alignment score for peptides with exact and non-exact matches. B. Number of target peptides grouped by their source organism.\n\nTaking the normalized alignment score of exact matches as a reference, we extracted 2019-nCoV peptides having score greater or equal to 4. As illustrated in Figure 1A, we observed 45 and 11 peptides having normalized alignment score ≥ 4 and ≥ 5 respectively (Figure 1A inset). The target peptides were originated from 10 different sources (Figure 1B) where a total 36 peptides were derived from strains associated to SARS CoV. Of interest, we also observed 7 hits having high sequence similarity to targets from Homo sapiens.\n\nIn order to investigate the extent to which the difference between the source (2019-nCoV) and target (IEDB) peptides influences the immunogenicity of the source peptides we used a recently published immunogenicity model5 to predict and compare the immunogenicity between the source and target peptides (Data Table 24).\n\nWe could see a similar (close to identical) immunogenicity scores for a number of IEDB and 2019-nCov peptides especially for those with high immunogenicity scores (Figure 2). While all 48 can be potential targets, of particular interest were those having higher immunogenicity score than IEDB peptides. Here, we list 22 out of 48 2019-nCoV peptides that scored higher compared to their targets that have been characterized to be immunogenic (Table 2). In this list 15 (68%) 2019-nCov peptides have a score higher than 0.5 whereas only 11(50%) of IEDB get a score immunogenicity score greater than 0.5.\n\n2019-nCoV peptides having a high sequence similarity to immunogenic peptides and their targets were analysed for their immunogenicity potential by iPred algorithm.\n\nIt is worth noting that in general predicting immunogenicity of given a peptide is challenging and not a fully solved problem, and therefore current models for predicting immunogenicity are suboptimal. iPred is also not an exception. In fact, we could see that a substantial number of IEDB immunogenic peptides were scored < 0.5 (the threshold score used to classify immunogenic vs non-immunogenic). This led us to ask whether we can gather any other evidence of either immunogenicity or cross-reactivity.\n\nAs a complementary reciprocal approach, we conducted a de novo search of immunogenic peptides against the 2019-nCov proteome sequence. We scanned 9-mers from 2019-nCoV proteome with a window of 9 amino acids and step length of 1 amino acid (9613 in total). The immunogenicity of 9-mer peptides were predicted using iPred and MHC presentation scores were gauged using NetMHCpan 4.06 for various HLA types. In this task, we focused on haplotypes common in Chinese and European populations, which include HLA-A*02:01, HLA-A*01:01, HLA-B*07:02, HLA-B*40:01 and HLA-C*07:02 alleles (Data Table 34).\n\nFor different alleles, 0 denotes non-binding and 1 denotes binding predicted for specific HLA allele.\n\nBased on MHC presentation and immunogenicity prediction, we detected 5 peptides predicted to bind 4 different HLA alleles of which 2 had strong immunogenicity scores (Figure 3). For those 65 strong binders to 3 different HLA types, 39 had immunogenicity scores ≥ 0.5 (Table 3). Collectively this analysis suggests a number of 9-mer immunogenic candidates for further experimental validation.\n\nThe MHC binding was predicted for HLA-A*02:01, HLA-A*01:01, HLA-B*07:02, HLA-B*40:01 and HLA-C*07:02 alleles by NetMHCpan 4.0 and immunogenicity was predicted by iPred.\n\nWhile our de novo candidates are appealing shortlisted targets for experimental validation, it does not provide information about target T cell receptors (TCRs). We therefore set out to interrogate the possibility of cross reactivity with one well-studied TCR.\n\nT cell cross-reactivity has been instrumental for the T cell immunity against both tumor antigens and external pathogens. In that regard, a number of T cells have been extensively characterized including 1G4 CD8+ TCR, which is known to recognize the ‘SLLMWITQC’ peptide presented by HLA-A*02:01. We therefore set out to leverage the data from a recently published study7 and exploit the possibility of cross reactivity of this TCR to any 2019-nCoV peptide.\n\nHere, we scanned all 9-mers from the 2019-nCoV proteome (9613 peptides) with Binding, Activating and Killing Position Weight Matrices (PWM, see the method section) and associated each peptide with the geometric mean of these three assays as a measure of immunogenicity (Data Table 44). The distributions of binding, activation and killing scores along with their multiplicative score and geometric mean are illustrated in Figure 4. Based on geometric mean, we observed 20 2019-nCoV peptides with a score > 0.8 and 516 peptides > 0.7. The 9-mer peptides with geometric mean > 0.7 and positive HLA-A*02:01 binding prediction by NetMHCpan 4.0 are listed in Table 4.\n\nThe positional weight matrices were obtained from 7 and 9613 9-mers generated from 10 2019-nCoV ORFs were computed for their TCR recognition potential.\n\nWe further analysed the MHC binding propensities and gathered peptides not only predicted positive by NetMHCpan but also to have leucine (L) and valine (V) in anchor positions 2 and 9 respectively. This led to identification of 44 2019-nCoV peptides of which 2 peptides had immunogenicity score > 0.7 and 12 peptides > 0.6 (Table 5). Thus, here we provide the list of peptides that are potential targets for 1G4 TCR recognition for subjects with HLA-A02:01 haplotype.\n\nPeptides have geometric mean ≥ 0.6 and ≤ 0.7 (for those ≥ 0.7, refer to Table 4) by 1G4 TCR positional weight matrix and predicted positive for HLA-A*02:01 binding by NetMHCpan 4.0 (Rank = NetMHCpan rank).\n\n\nDiscussion\n\nIn this study we provide a profile of computationally predicted immunogenic peptides from 2019-nCoV for functional validation and potential vaccine developments. We are fully aware that an effective vaccine development will require a very thorough investigation of immune correlates to 2019-nCoV. However, due to the emergency and severity of the outbreak as well as the lack of access to samples from infected subjects, such approaches would not serve the urgency. Therefore, computational prediction is instrumental for guiding biologists towards a quick and cost-effective solution to prevent the spread and ultimately help eliminate the infection from the individuals.\n\nWith a rising global concern of novel coronavirus outbreak, numerous research groups have started to investigate and publish their findings. At the time of preparing this manuscript, we became aware of a similar study conducted in comparing 2019-nCoV proteome with SARS CoV immunogenic peptides8. Our in silico approach takes the search beyond presenting only common immunogenic peptide between SARS and 2019-nCoV and provides the experimental community with a more comprehensive list including de novo and cross reactive candidates. On the other hand, considering the fact that two studies have been accomplished independently with distinct approaches, this serves to demonstrate a high level of confidence in reproducing the results. Reproducibility of computational prediction is always of high importance and becomes even more significant under urgent scenarios as of this outbreak.\n\nOur study also suggests the need for further efforts to develop accurate predictive models and algorithms for the characterization of immunogenic peptides.\n\nIn this study, we provide potential immunogenic peptides from 2019-nCoV for vaccine targets that i) have been characterized immunogenic by previous studies on SARS CoV, ii) have high degree of similarity with immunogenic SARS CoV peptides and iii) are predicted immunogenic by combination of NetMHCpan and iPred/1G4 TCR positional weight matrices. Given the limited time and resources, our work serves as a guide to save time and cost for further experimental validation.\n\n\nMethod\n\n2019-nCoV open reading frame sequences were downloaded from NCBI (MN908947.3). All sequences subjected for analysis are deposited in GitHub repository.\n\nAll subsequent analyses have been conducted in R 3.6.1.\n\nThe sequence similarity between 2019-nCoV open reading frames and previously characterized immunogenic peptides in IEDB was analysed by local alignment using R ‘pairwiseAlignment’ function from Biostrings v2.40.2 package. The local alignment utilized BLOSUM62 matrix, gapOpening of 5 and gapExtension of 5. The alignment score was normalized by length of target peptides.\n\nWe have used iPred5 to predict immunogenicity of each given peptide. Briefly, iPred employs peptides’ length and physicochemical properties of amino acids modelled by sums of ten Kidera factors and associates a score to each peptide reflecting its likelihood of recognition by a T cell.\n\nIn order to predict peptide binding to MHC we used NetMHCpan V46. This version of NetMHCpan that comes with a number of improvements, incorporate both eluted ligand and peptide binding affinity data into a neural network model to predict MHC presentation of each given peptide.\n\nTo gauge the level of 1G4 TCR cross-reactivity to list of 2019-nCoV virus, we have leveraged the data from a recently published study7. 1G4 or NY-ESO-1-specific TCR is a very well-studied and clinically efficacious TCR which recognize the peptide ‘SLLMWITQC’ presented by HLA-A*02:01. Karapetyan et al. have recently provided data from three experimental assays reflecting Binding, Activating and Killing upon each mutation at each position of all possible 9-mers using these three datasets. In a similar way to the original paper, we trained three Position Weight Matrices named B, A and K respectively from Binding, Activating and Killing assay. We defined the cross-reactivity score of a given 9-mer sequence as the geometric mean of B, A and K.\n\nWe then scanned 2019-nCoV virus protein sequence with each of B, A and K PWMs and associated each of 9613 9-mers with a cross reactivity score. At the same we utilized NetMHCpan and associated each 9-mer with its presentation score. Our final list of cross-reactive candidate peptides were those with a cross-reactivity sore >= 0.8 and reported as strong binders from NetMHCpan and have ‘L’ and ‘V’ amino acids at anchor positions. The custom R codes are accessible from GitHub repository (see software availability4).\n\n\nSoftware availability\n\nReplication code: https://github.com/ChloeHJ/Vaccine-target-for-2019-nCoV.git\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.36769084\n\nLicense: Creative Commons Attribution 4.0 International\n\n\nData availability\n\n2019-nCoV open reading frame sequences were downloaded from NCBI (MN908947.3).\n\nZenodo: In silico identification of vaccine targets for 2019-nCoV (Data tables). http://doi.org/10.5281/zenodo.36768869\n\nThis project contains the following underlying data:\n\n– Table1 nCoV peptides having exact match with immunogenic SARS CoV peptides.xlsx (Table of nCoV peptides having exact match with immunogenic SARS CoV peptides)\n\n– Table2 nCoV peptides with high sequence similarity with immunogenic IEDB peptides.csv (Table of peptides with high sequence similarity with immunogenic IEDB peptides)\n\n– Table3 de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and iPred.csv (Table of results of de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and iPred)\n\n– Table4 de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and PWM.xlsx (Table of results of de novo search on 9-mer nCoV for immunogenic peptides by NetMHCpan and PWM)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe acknowledge further appreciate assistance and computing support from Unit and WIMM Centre for Computational Biology at MRC Weatherall Institute of Molecular Medicine. We thank G. Napolitani and M. Salio for insightful discussions about the project.\n\n\nReferences\n\nWorld Health Organization: 2019-NCoV Outbreak Is an Emergency of International Concern. World Health Organization, 31 Jan. 2020. Reference Source\n\nKock RA, Karesh WB, Veas F, et al.: 2019-nCoV in context: lessons learned? Lancet Planet Health. 2020; pii: S2542-5196(20)30035-8. PubMed Abstract | Publisher Full Text\n\nVita R, Mahajan S, Overton JA, et al.: The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Res. 2019; 47(D1): D339–D343. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee CH, Koohy H: In silico identification of vaccine targets for 2019-nCoV (Custom code). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3676908\n\nPogorelyy MV, Fedorova AD, McLaren JE, et al.: Exploring the pre-immune landscape of antigen-specific T cells. Genome Med. 2018; 10(1): 68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJurtz V, Paul S, Andreatta M, et al.: NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J Immunol. 2017; 199(9): 3360–3368. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarapetyan AR, Chaipan C, Winkelbach K, et al.: TCR Fingerprinting and Off-Target Peptide Identification. Front Immunol. 2019; 10: 2501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhmed SF, Quadeer AA, McKay MR, et al.: Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. 2020. Publisher Full Text\n\nLee CH, Koohy H: In silico identification of vaccine targets for 2019-nCoV (Data tables). [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3676886"
}
|
[
{
"id": "60504",
"date": "05 Mar 2020",
"name": "Katalin A. Wilkinson",
"expertise": [
"Reviewer Expertise T cell immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important manuscript identifying potential vaccine targets for 2019-nCoV, using computational prediction. In the absence of patient samples, such approaches are instrumental to guide towards quick and efficient identification of vaccine candidates. The authors used 10 open reading frame sequences of 2019-nCoV deposited at NCBI and conducted sequence alignment against immunogenic peptides deposited in the Immune Epitope Database and Analysis Resource (IEDB) database. They identified 28 peptides with sequences matching exactly to severe acute respiratory syndrome-related coronavirus (SARS CoV), that have previously been characterised as immunogenic by T cell assays. These findings are very promising and have the added benefit of potentially developing a vaccine against both SARS and COVID-19.\n\nAdditional peptides were identified to most likely bind common Chinese and European HLA alleles and have high immunogenicity potential. The authors provide a shortlist of peptides as potential vaccine candidates. While this manuscript presents a good model for identifying such targets, a comment should be included in the discussion about the necessity of expanding the analysis to include wider HLA allele types, considering that the virus is likely to spread worldwide.\nMinor comment: Please explain the methods and analysis in greater details, including the following terms:\n‘Towards target peptide length > 3’.\n\n‘Normalized alignment scores’ (and their scale, such as the significance of 4 for the data presented in Figure 1).\n\nThe importance of leucine (L) and valine (V) in anchor positions for MHC binding (for data presented in Table 5).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": [
{
"c_id": "5404",
"date": "07 Apr 2020",
"name": "Hashem Koohy",
"role": "Author Response",
"response": "Dear Prof Wilkinson, Thank you so much for your time and valuable suggestions. In our new and revised version we have taken your suggestions into consideration. Best regards Hashem Koohy"
}
]
},
{
"id": "60502",
"date": "31 Mar 2020",
"name": "Raffaele De Palma",
"expertise": [
"Reviewer Expertise T cell responses in Health and Diseases. Modulation of Immune Responses. Clinical Immunology. Vaccines"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors use an in silico approach to identify antigenic peptides derived from 2019-nCoV. First, they screened ten open reading frame of 2019-nCoV sequence used the IEDB database, finding a series of peptides potentially immunogenic. The first piece of data relies on the identification of 28 peptides that were exactly matching SARS CoV peptides. Moreover, using combinatory approaches, modelling HLA and TCR binding, they identified 13 peptides potentially able to bind a given TCR in HLA A2 restriction fashion, and cutting a list of peptides able to bind several HLA alleles and characterizing several peptides that may be de novo candidates or crossreactive peptides to be used either to study immune response to 2019-nCoV or to set a vaccine.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-145
|
https://f1000research.com/articles/8-2072/v1
|
10 Dec 19
|
{
"type": "Research Article",
"title": "Widespread use of the “ascidian” mitochondrial genetic code in tunicates",
"authors": [
"Julien Pichon",
"Nicholas M. Luscombe",
"Charles Plessy",
"Julien Pichon",
"Nicholas M. Luscombe"
],
"abstract": "Background: Ascidians, a tunicate class, use a mitochondrial genetic code that is distinct from vertebrates and other invertebrates. Though it has been used to translate the coding sequences from other tunicate species on a case-by-case basis, it is has not been investigated whether this can be done systematically. This is an important because a) some tunicate mitochondrial sequences are currently translated with the invertebrate code by repositories such as NCBI GenBank, and b) uncertainties about the genetic code to use can complicate or introduce errors in phylogenetic studies based on translated mitochondrial protein sequences. Methods: We collected publicly available nucleotide sequences for non-ascidian tunicates including appendicularians such as Oikopleura dioica, translated them using the ascidian mitochondrial code, and built multiple sequence alignments covering all tunicate classes. Results: All tunicates studied here appear to translate AGR codons to glycine instead of serine (invertebrates) or as a stop codon (vertebrates), as initially described in ascidians. Among Oikopleuridae, we suggest further possible changes in the use of the ATA (Ile → Met) and TGA (Trp → Arg) codons. Conclusions: We recommend using the ascidian mitochondrial code in automatic translation pipelines of mitochondrial sequences for all tunicates. Further investigation is required for additional species-specific differences.",
"keywords": [
"Tunicate",
"Oikopleura",
"Genetic code",
"Mitochondria",
"Cytochrome oxidase subunit I"
],
"content": "Introduction\n\nTunicates are marine animals that have acquired the capacity to produce cellulose by horizontal gene transfer approximately 500 million years ago (Matthysse et al., 2004; Nakashima et al., 2004). Together with vertebrates and cephalochordates, they belong to the chordate phylum, in which they share morphological features such as a muscular tail during larval stages. Phylogenetic studies place the tunicates as the closest living relatives of vertebrates (Delsuc et al., 2006). Tunicates can be subdivided in three classes: Thaliacea (free-swimming colonial species, for instance salps or dolioids), Appendicularia (free-swimming solitary species with an adult morphologically similar to the larval stage of other tunicates), and Ascidiacea (attached to solid substrates in their adult stage, for instance sea squirts). The relationship between these classes and therefore their mono- or paraphyly has been revised multiple times. For instance the 18S rRNA analysis of Stach & Turbeville (2002) nested Appendicularia within Ascidiacea, but more recently Delsuc et al. (2018) placed them as sister groups using a multigene approach. The paraphyly of Ascidiacea is now widely accepted, as the above studies and others demonstrated that they contain the Thaliacea.\n\nMitochondrial genomes undergo major changes at the geological time scale due to their small size and clonal reproduction, including changes to their genetic code (Osawa et al., 1992). The first evidence that ascidians use a specific mitochondrial genetic code stemmed from observations that the cytochrome c oxidase subunit 1 (Cox1) sequence from Halocynthia roretzi (Yokobori et al., 1993) and the Cox3 sequence of Pyura stolonifera (Durrheim et al., 1993) are interrupted by stop codons if translated using the vertebrate mitochondrial code. Reassignment of AGR codons to glycine was later confirmed by the discovery of a glycine (Gly) tRNA in the H. roretzi genome (Yokobori et al., 1999) and by the sequencing of its anticodon (U*CU) (Kondow et al., 1999). Apart from the AGR codons, the ascidian code is similar to the vertebrate and the invertebrate ones, with ATA assigned to methionine (Met) and TGA to tryptophan (Trp) (Yokobori et al., 1993).\n\nThis genetic code is known as the “ascidian” genetic code; however, it is also used by non-ascidian tunicates, such as the thaliacean Doliolum nationalis (Yokobori et al., 2005). The possibility that this genetic code emerged earlier than tunicates was raised by the study of partial genome sequences of Branchiostoma lanceolatum (Delarbre et al., 1997) leading to the proposition that AGR might encode Gly in cephalochordates. While this seemed to be supported by the discovery of a putative TCT (Gly) tRNA in the full mitochondrial genome of B. lanceolatum (Spruyt et al., 1998), this hypothesis was later ruled out by an analysis of the related amphioxus Branchiostoma floridae (Boore et al., 1999), and has not been reconsidered since. Finally, studies on the appendicularian branch showed compatibility between the mitochondrial sequence of Oikopleura dioica and the ascidian code (Denoeud et al., 2010). Nevertheless, support for compatibility was not demonstrated explicitly for the ATA and TGA codons and the mitochondrial sequence of O. dioica were not released in International Nucleotide Sequence Database Collaborations (INSDC) databanks.\n\nCox1 is the most conserved mitochondrial protein. Although no mitochondrial genome has been fully sequenced yet for appendicularians, partial Cox1 sequences are present in the INSDC databanks for Oikopleuridae. Sakaguchi et al. (2017) reported that all Oikopleura mitochondrial sequences (AY116609–AY116611 and KF977307) may be contaminations from bacteria or cnidarians, and provided partial sequences for Oikopleura longicauda in the same study. Partial mitochondrial sequences were published for Bathochordaeus and Mesochordaeus species by Sherlock et al. (2017). In addition, Naville et al. (2019) recently published draft genome for several appendicularian species. Therefore, to assess whether the ascidian mitochondrial code is used across the whole tunicate subphylum, we took advantage of these public data and prepared a curated alignment of Cox1 sequences comprising representatives of the major tunicate branches, to study the consensus sequences at conserved residues.\n\n\nMethods\n\nWe identified Cox1 and Cytochrome b (Cob) gene sequences for Oikopleura longicauda, Mesochordaeus erythrocephalus and Bathochordaeus stygius by screening published genome assemblies (Naville et al., 2019) with the partial Cox1 sequence of O. longicauda LC222754.1 (Sakaguchi et al., 2017) using tblastn and the ascidian mitochondrial code (-db_gencode=13) (Gertz et al., 2006). Mitochondrial genome sequences were then translated using the cons and getorf commands from EMBOSS (Rice et al., 2000), using the ascidian mitochondrial code.\n\nWe identified the circular contig SCLD01101138.1 (length: 10,324 nt) as a potential mitochondrial genome, and translated Cox1 from position 4530 to 6230. We also translated Cob from 3697 to 4668.\n\nWe translated Cox1 in contig SCLF01725989.1 (length 7,034 nt) on reverse strand from position 1792 to 272. Using the same method with O. longicauda’s Cob sequence as a bait, we also recovered a Cob sequence from contig SCLF01109548.1 (length 5,010 nt), reverse strand, 1604 to 2590.\n\nWe used the consensus of the published B. stygius Cox1 sequences KX599267.1 to KX599281.1 from GenBank (Sherlock et al., 2017), to screen the genome and scaffold SCLE01415711.1 (length 10,388 nt) gave a perfect hit. We translated Cox1 from position 8054 to 6522 on the reverse strand, and a partial Cob sequence from scaffold SCLE01415711.1 (2319 to 2963, reverse strand). We also found a second fragment aligning well with C-terminal sequences between positions 2373 and 1978, but we did not include it due to the difficulty of resolving the overlap between both fragments. When screening with the M. erythrocephalus Cox1 sequence recovered above, we found that another scaffold SCLE01416475.1 gave a perfect hit, hinting at a possible contamination.\n\nTo assemble a Cox1 sequence in O. dioica, we downloaded expressed sequence tags (file 10_ESTall.txt) from Oikobase (Danks et al., 2012) and extracted hits matching the O. longicauda sequence using tblastn (see above). We then aligned and visualised the hits using Clustal Omega (Sievers et al., 2011) and SeaView (Gouy et al., 2009), filtering out those too short or introducing gap columns. Inspection of the alignment let us notice three possible haplotypes. We generated a consensus for each of them, translated them (see above) and trimmed the proteins sequences in order to match the length of the other reference sequences in the alignment. All variants found between the haplotypes were synonymous codons. We used the same methodology to generate a consensus for Cob and translate it.\n\nBathochordaeus charon KT881544.1 ORF2 translated with ascidian code; Bathochordaeus stygius: SCLE01415711.1[8054:6522] translated with ascidian code; Branchiostoma lanceolatum:BAD93656.1; Caenorhabditis elegans: NP_006961.1; Ciona intestinalis: CAL23359.2; Clavelina oblonga: YP_009029840.1; Doliolum nationalis: BAD86512.1; Halocynthia roretzi: NP_038239.1; Mesochordaeus erythrocephalus: SCLF01725989.1[1915:260] translated with ascidian code; Mus musculus: NP_904330.1; Oikopleura dioica: consensus of Oikobase contigs (see file 10_ESTall.txt) KT0AAA24YA11, KT0AAA22YO17, KT0AAA22YO04, KT0AAA13YK14, KT0AAA18YK22, KT0AAA16YP04, KT0AAA13YE23, KT0AAA8YH10, KT0AAA4YK01, KT0AAA24YE23, KT0AAA18YO18, KT0AAA3YP19, KT0AAA10YF12; O. longicauda: SCLD01101138.1[4678:6230] translated with ascidian code; Salpa thompsoni: BBB04277.1.\n\nBathochordaeus stygius: SCLE01415711.1[2963:2319] translated with ascidian code; Branchiostoma lanceolatum: BAD93666.1; Caenorhabditis elegans: NP_006958.1; Ciona intestinalis: CAL23352.2; Clavelina oblonga: YP_009029843.1; Doliolum nationalis: BAD86520.1; Halocynthia roretzi: NP_038246.1; Mesochordaeus erythrocephalus: SCLF01109548.1[1604:2590] translated with ascidian code; Mus musculus: NP_904340.1; Oikopleura dioica: consensus of Oikobase contigs KT0AAA23YJ17, KT0AAA16YJ22, KT0AAA17YO14, KT0AAA10YI15, KT0AAA18YI18, KT0AAA11YF07, KT0AAA10YG05, KT0AAA1YH02, KT0AAA12YH10, KT0AAA12YC07, KT0AAA12YC07, KT0AAA18YM15 (see file 10_ESTall.txt); O. longicauda: SCLD01101138.1[3697:4668] translated with ascidian code; Salpa thompsoni: BBB04269.1.\n\nTranslated Cox1 and Cob sequences were aligned using Clustal Omega (Sievers et al., 2011) and SeaView (Gouy et al., 2009). The alignments were post-processed using the showalign -show=n command of EMBOSS (Rice et al., 2000) to show the differences to the inferred consensus. Graphical processing of the alignments were performed with Jalview (Waterhouse et al., 2009). The codon sequences encoding Cox1 and Cob of the tunicate species were then added aligned to the corresponding amino-acid (three lines per species, see Extended data (Plessy & Pichon, 2019)) and then the text files were transposed, so that each line would correspond to a single position in the alignment, and interrogated with custom Unix commands to compute the tables presented in this manuscript.\n\n\nResults\n\nWe selected species according to sequence availability and to ensure coverage of the tunicate subphylum in a way that stays broad under the various hypotheses of monophyly or paraphyly for its major groups. For ascidians, we have included the phlebobranchian Ciona intestinalis, the aplousobranchian Clavelina oblonga and the pyurid stolidobranchian Halocynthia roretzi. For thaliaceans, we selected Doliolum nationalis and Salpa thompsoni. For appendicularians we selected Oikopleura dioica, Oikopleura longicauda, Bathochordaeus stygius and Mesochordaeus erythrocephalus. We ensured that all tunicate sequences were translated with the ascidian mitochondrial genetic code. Lastly, we included outgroup sequences from Caenorhabditis elegans and Branchiostoma lanceolatum (invertebrate mitochondrial code) and from Mus musculus (vertebrate mitochondrial code) to better highlight conserved amino acid positions. In Figure 1, we illustrate the relation between these species based on the phylogeny of Naville et al. (2019) for appendicularians and of Delsuc et al. (2018) for the other tunicates. We prepared Cox1 sequences from the selected species using mitochondrial genomes (for ascidians, thaliaceans, and outgroups), from draft genomes in which we found a putative mitochondrial contig after screening with a partial or a related Cox1 sequence (for O. longicauda, B. stygius, and M. erythrocephalus) and from EST sequences (for O. dioica). We aligned the translated Cox1 and Cob sequences (Figure 2 and Figure 3) and inspected the positions where all species use the same amino acid. Conserved glycines supported the use of AGR codons across the whole tunicate clade. We confirmed this observation with Cob sequences obtained with the same method.\n\nDifferent branch colors indicate different mitochondrial genetic codes. Codon assignments with an equal sign indicate how the nucleotide sequences were translated. Codon assignments with a question mark indicate a possible finding, but were not used for translation. Ascidians, in which the AGR to Gly codon reassignment was initially discovered, are highlighted among the tunicates. Right: codon sequence of Cox1 genes on positions where proposed changes of genetic code would make all species use the same amino acid.\n\nWhite stars indicate conserved cysteines when at least one tunicate uses an AGR codon. Black stars indicate positions suggesting a different genetic code.\n\nWhite stars indicate conserved cysteines when at least one tunicate uses an AGR codon. Black stars indicate positions suggesting a different genetic code.\n\nWe then searched for positions where a single tunicate species differed from the other sequences with the same replacement amino acid more than once. We found multiple cases of methionine being replaced by isoleucine and arginine replaced by tryptophan in O. longicauda and B. stygius (Figure 2). Given their phylogenetic proximity, we grouped the two species in the analysis below and we calculated the number of mismatches to the other sequences. We redefined a position as “conserved” if there is at most one mismatch from one sequence to the others.\n\nM. erythrocephalus does not seem to use ATA codons and O. longicauda and B. stygius use ATA codons at positions where all other species had an isoleucine (Ile) (Table 1 and Table 2). In the ancestral invertebrate mitochondrial code and the sister vertebrate code, ATA encodes Met. Although Met and Ile both have hydrophobic side chains that often can substitute for each other, this also suggests a change of the genetic code. Evidence for this is that 1) non-appendicularian species do not display ATA codons at positions where all other species encode Ile; 2) the change would be parsimonious as O. longicauda, B. stygius and M. erythrocephalus are more closely related to each other than to O. dioica (Naville et al., 2019); and 3) these three species never have ATA codons at positions where Met is conserved in every species (in contrast to O. dioica). Furthermore, reversion of the ATA codon to Ile have occurred in other branches of the tree of Life, for instance in echinoderms (Jacobs et al., 1988). Finally, inspection of a partial Cox1 sequence of the related Bathochordaeus charon (KT881544.1) provided one extra instance of an ATA codon at a conserved Ile position.\n\nThe TGA codon is known to encode tryptophan (Trp) in vertebrate, invertebrate and ascidian mitochondria (Fox, 1979). We found that B. stygius uses TGA at positions where all other species would encode Arg (Table 3 and Table 4). This is surprising as these two amino acids are unlikely to functionally substitute for each other. O. longicauda does not use TGA codons, and M. erythrocephalus does not use TGA at conserved Arg, although it is found at a position where all other species encode for Arg except C. elegans which encodes lysine, the other positively charged amino-acid. This again suggests a possible change of genetic code, although the numbers are currently too small to draw a solid conclusion.\n\n\nDiscussion\n\nWe extracted Cox1 and Cob sequences of four different appendicularians from public databases. As a nucleotide sequence, Cox1 might be useful for mining databases of molecular barcodes sequenced from the environment, or for studies of population diversity within a species. As a protein sequence, Cox1 might be useful for refining the phylogeny of appendicularians. However, a translation code needs to be chosen.\n\nOur alignments of tunicate Cox1 and Cob protein sequences support the view that all tunicates translate AGR codons as Gly (although this conclusion might be limited by the lack of coverage for the Kowalevskiidae and Fritillariidae families). While our analysis suggests that the last common ancestor of the tunicates used the “ascidian” code, it is not possible to conclude that all contemporary tunicates still do, as we found discrepancies on other conserved residues that could be explained by a genetic code change of ATA and TGA codons within a sub-clade of the appendicularians containing M. erythrocephalus, O. longicauda and B. stygius.\n\nThe “ascidian” genetic code is table number 13 in the NCBI protein database, where it is used to translate sequences from ascidians and non-ascidian tunicates, for instance D. nationalis. However for appendicularians, the NCBI currently applies the invertebrate table (number 5). This has the consequences of turning Gly to Ser at functionally important positions. Therefore, the ascidian is probably a more appropriate default. At present, it is unclear whether some appendicularians have additional changes; however, the accurate translation of AGR codons to Gly would nonetheless reduce the amount of error in translated protein sequences.\n\nTo confirm a change of genetic code, it is necessary to detect corresponding changes in the respective tRNAs. This beyond reach for the present study because the mitochondrial genomic sequences that we used are extracted from draft genome sequences that may be incomplete, or even contain contaminations (see B. stygius in the Methods section). As a result, we also cannot entirely rule out the possibility that we have examined pseudogenes, although the high conservation found in the alignments suggest this in unlikely. For all these reasons, it is necessary to sequence full-length mitochondrial genomes from appendicularians.\n\n\nConclusions\n\nOur alignments of translated mitochondrial sequences suggest that the last common ancestor of living tunicates may have already used the “ascidian” genetic code. Thus, we recommend the use of that code instead of the “invertebrate” one for all tunicates in automatic translation pipelines, with the caveat that additional changes might be found in appendicularians. This observation is a reminder that in biology, exception is the rule, and that each time a mitochondrial sequence is extracted from a species for the first time, it is important to carefully examine its genetic code.\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: Aligned Cox1 and Cob sequences from Oikopleura dioica and other tunicates. https://doi.org/10.5281/zenodo.3490310 (Plessy & Pichon, 2019).\n\nThis project contains alignment files and descriptions of how the files were generated.\n\nExtended data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe thank the OIST’s Scientific Computing & Data Analysis Section for their support, and Ferdinand Marlétaz for critical comments on our manuscript.\n\n\nReferences\n\nBoore JL, Daehler LL, Brown WM: Complete sequence, gene arrangement, and genetic code of mitochondrial DNA of the cephalochordate Branchiostoma floridae (Amphioxus). Mol Biol Evol. 1999; 16(3): 410–418. PubMed Abstract | Publisher Full Text\n\nDanks G, Campsteijn C, Parida M, et al.: OikoBase: a genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica. Nucleic Acids Res. 2012; 41(Database issue): D845–D853. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDelarbre C, Barriel V, Tillier S, et al.: The main features of the craniate mitochondrial DNA between the ND1 and the COI genes were established in the common ancestor with the lancelet. Mol Biol Evol. 1997; 14(8): 807–813. PubMed Abstract | Publisher Full Text\n\nDelsuc F, Brinkmann H, Chourrout D, et al.: Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature. 2006; 439(7079): 965–968. PubMed Abstract | Publisher Full Text\n\nDelsuc F, Philippe H, Tsagkogeorga G, et al.: A phylogenomic framework and timescale for comparative studies of tunicates. BMC Biol. 2018; 16(1): 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDenoeud F, Henriet S, Mungpakdee S, et al.: Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate. Science. 2010; 330(6009): 1381–1385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDurrheim GA, Corfield VA, Harley EH, et al.: Nucleotide sequence of cytochrome oxidase (subunit III) from the mitochondrion of the tunicate Pyura stolonifera: evidence that AGR encodes glycine. Nucleic Acids Res. 1993; 21(15): 3587–3588. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFox TD: Five TGA \"stop\" codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II. Proc Natl Acad Sci U S A. 1979; 76(12): 6534–6538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGertz EM, Yu YK, Agarwala R, et al.: Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST. BMC Biol. 2006; 4: 41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGouy M, Guindon S, Gascuel O: SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2009; 27(2): 221–224. PubMed Abstract | Publisher Full Text\n\nJacobs HT, Elliott DJ, Math VB, et al.: Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J Mol Biol. 1988; 202(2): 185–217. PubMed Abstract | Publisher Full Text\n\nKondow A, Suzuki T, Yokobori S, et al.: An extra tRNAGly(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine. Nucleic Acids Res. 1999; 27(12): 2554–2559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatthysse AG, Deschet K, Williams M, et al.: A functional cellulose synthase from ascidian epidermis. Proc Natl Acad Sci U S A. 2004; 101(4): 986–991. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakashima K, Yamada L, Satou Y, et al.: The evolutionary origin of animal cellulose synthase. Dev Genes Evol. 2004; 214(2): 81–88. PubMed Abstract | Publisher Full Text\n\nNaville M, Henriet S, Warren I, et al.: Massive Changes of Genome Size Driven by Expansions of Non-autonomous Transposable Elements. Curr Biol. 2019; 29(7): 1161–1168.e6. PubMed Abstract | Publisher Full Text\n\nOsawa S, Jukes TH, Watanabe K, et al.: Recent evidence for evolution of the genetic code. Microbiol Rev. 1992; 56(1): 229–264. PubMed Abstract | Free Full Text\n\nPlessy C, Pichon J: Aligned Cox1 and Cob sequences from Oikopleura dioica and other tunicates. 2019. http://www.doi.org/10.5281/zenodo.3490310\n\nRice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000; 16(6): 276–277. PubMed Abstract | Publisher Full Text\n\nSakaguchi SO, Ikuta T, Ogawa G, et al.: Morphological identity of a taxonomically unassigned cytochrome c oxidase subunit i sequence from stomach contents of juvenile chum salmon determined using polymerase chain reaction. Fish Sci. 2017; 83(5): 757–765. Publisher Full Text\n\nSherlock RE, Walz KR, Schlining KL, et al.: Morphology, ecology, and molecular biology of a new species of giant larvacean in the eastern North Pacific: Bathochordaeus mcnutti sp. nov. Mar Biol. 2017; 164(1): 20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSievers F, Wilm A, Dineen D, et al.: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7: 539. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpruyt N, Delarbre C, Gachelin G, et al.: Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates. Nucleic Acids Res. 1998; 26(13): 3279–3285. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStach T, Turbeville JM: Phylogeny of Tunicata inferred from molecular and morphological characters. Mol Phylogenet Evol. 2002; 25(3): 408–428. PubMed Abstract | Publisher Full Text\n\nWaterhouse AM, Procter JB, Martin DM, et al.: Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009; 25(9): 1189–1191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYokobori SI, Oshima T, Wada H: Complete nucleotide sequence of the mitochondrial genome of Doliolum nationalis with implications for evolution of urochordates. Mol Phylogenet Evol. 2005; 34(2): 273–283. PubMed Abstract | Publisher Full Text\n\nYokobori S, Ueda T, Feldmaier-Fuchs G, et al.: Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretzi (Chordata, Urochordata). Genetics. 1999; 153(4): 1851–1862. PubMed Abstract | Free Full Text\n\nYokobori SI, Ueda T, Watanabe K: Codons AGA and AGG are read as glycine in ascidian mitochondria. J Mol Evol. 1993; 36(1): 1–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "57710",
"date": "13 Jan 2020",
"name": "Patrick Lemaire",
"expertise": [
"Reviewer Expertise Tunicate embryology and genomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this short article Pichon and colleagues use publicly available molecular datasets, mostly partial mitochondrial genomes, to re-explore the mitochondrial genetic code across tunicates, based on the analysis of the Cox1 and Cob genes.\nTheir data suggest that the AGR codon was already translated into Glycine in the last common ancestor of tunicates and that additional changes may have occured in some Oikopleuridae at least. The work is important because it shows that all tunicates, including appendicularians should be associated in Genbank to the \"ascidian\" genetic code (Table 13).\nA limitation of the work, which the authors acknowledge, is that they could not identify the corresponding tRNAs in appendicularians. They therefore call for the sequencing of full-length mitochondrial genomes for appendicularians.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "59671",
"date": "26 Feb 2020",
"name": "Yuanning Li",
"expertise": [
"Reviewer Expertise Evolutionary genomics and phylogenetics in marine invertebrates."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPichon et al. conducted a study by comparing two mt genes across tunicates and recovered conserved tunicate-specific mt codon usage and potential Oikopleuridae-codon. The writing of the manuscript is clear and easy to follow. The methods are also valid and the findings should be interesting and important to the field. However, there are a few things I suggest to incorporate in the current draft.\nIn the introduction, the authors mainly discussed the tunicate genetic codon, but we already know there are more codon changes in deuterostomes (e.g. Hemichordates contain two mt genetic codons). So it would be better to incorporate this part of the introduction to make sure readers understand there are many mt codon changes within deuterostomes.\n\nThe authors also briefly discussed the possible change of tRNA structures responsible for codon change. Is it possible to extract tRNA sequences from available tunicate transcriptomic data and compare them to the existing ones? I am also fine with this if that is beyond the scope of this manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5344",
"date": "14 Apr 2020",
"name": "Charles Plessy",
"role": "Author Response",
"response": "We thank the reviewer for their 2 suggestions. 1) “To make sure readers understand there are many mt codon changes within deuterostomes”, we are adding the following text and references to our introduction: In animals, alternative genetic codes have first been found in large clades, for instance echinoderms (Himeno et al., 1987) and hemichordates (Castresana et al., 1998), but more recent works underline the presence of changes deeper in the phylogenetic tree, for instance within nematodes (Jacob et al., 2009) and within hemichordates (Li et al., 2019). 2) On whether it is “possible to extract tRNA sequences from available tunicate transcriptomic data”: as absence of evidence is not evidence for absence, our standpoint is that a rigorous analysis using a reference mitochondrial genome will be preferable. We hope that our methods section, that points at genomic scaffolds that are potential drafts of mitochondrial genomes, will be useful to the researchers interested in pursuing this direction."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2072
|
https://f1000research.com/articles/7-1907/v1
|
07 Dec 18
|
{
"type": "Research Article",
"title": "Usual care for mental health problems in children with epilepsy: A cohort study",
"authors": [
"Alice Welch",
"Roz Shafran",
"Isobel Heyman",
"Anna Coughtrey",
"Sophie Bennett",
"Alice Welch",
"Roz Shafran",
"Isobel Heyman",
"Anna Coughtrey"
],
"abstract": "Background: Epilepsy is one of the most common chronic paediatric conditions. Children and young people with epilepsy are at a significantly higher risk of developing mental health problems relative to the general population, yet the majority of these problems are unrecognised and under-treated in clinical practice. Although there is little epilepsy-specific guidance as to what interventions to use, researchers suggest there is no reason why clinicians should not be using the evidence base. Given the poor prognosis of untreated mental health difficulties, this cohort study sought to identify what psychological treatment young people with epilepsy with mental health needs receive in routine practice. Methods: Participants were children and young people aged 3 to 18 attending paediatric neurology clinics. The parents of those children who met threshold for impairing symptoms on the Strengths and Difficulties questionnaire were asked to complete the Development and Well-being Assessment (DAWBA), an online clinical assessment designed to generate psychiatric diagnoses. Participants who met clinical threshold for a disorder according to the DAWBA were provided with a bespoke measure asking questions regarding their experience with treatment for mental health support. Results: 16 of the 46 parents who completed the DAWBA reported that they had experienced previous or current support for their child’s mental health difficulties. The mental health support offered to families was highly variable, inadequate and often not clearly compliant with existing UK National Institute for Health and Clinical Excellence (NICE) guidelines for mental health treatment in children and young people. Conclusions: The present study demonstrates the inconsistency and inadequacy of mental health provision for children and young people with epilepsy. Future work should explore reasons for the treatments offered failing to adhere to existing guidance for mental health difficulties in children, as well as possible solutions to this.",
"keywords": [
"Epilepsy",
"Mental health",
"CAMHS",
"Paediatric"
],
"content": "Introduction\n\nEpilepsy is one of the most common paediatric neurological conditions in childhood (Hirtz et al., 2007). Children and young people with epilepsy have a greatly elevated risk of developing mental health difficulties relative to that of the general population (e.g. Rodenburg et al., 2005) and of children with other Long Term Conditions, such as diabetes (e.g. Davies Heyman & Goodman et al., 2003). Presence of psychiatric comorbidities may explain lower ratings in Health-related Quality of Life than seizure or demographic variables (Baca et al., 2011; Stevanovic et al., 2011). Further, poor mental health in children may contribute to a greater severity of physical illness (Miller et al., 2009), for example the presence of a mental health disorder has been linked to a greater frequency of seizures (de Araujo Filho & Yacubian, 2013). As a result, the UK National Institute for Health and Clinical Excellence (NICE) guidelines recommend that the psychological needs of children with epilepsy should be considered as part of routine care (NICE, 2012, p.52).\n\nDespite the recommendations from NICE, there is a lack of research into the optimal psychological treatment of mental health disorders in children with epilepsy (Jones, 2014), which means that there is little direction as to the types of assessments and interventions that should be used to identify and treat mental health difficulties in this group. The mental health difficulties most commonly seen in children with epilepsy are also those seen most commonly in children without epilepsy (for example anxiety, depression, disruptive behaviour, autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD); (Davies et al., 2003) and there is a wealth of research supporting the use of evidence based treatments for these disorders in children without epilepsy (e.g. Weisz et al., 2013). For example, UK NICE guidelines (2013) recommend that, in children and young people without epilepsy who have mental health needs, those with disruptive behaviour disorders should be given approximately ten hourly sessions of a behavioural parenting intervention as a first-line treatment and those with social anxiety should be given 8–12 sessions of cognitive behaviour therapy of 45 minutes duration (NICE, 2013). Given that cognitive behaviour therapy (CBT) and behavioural parenting interventions have been shown to work across a number of different populations, including those with Intellectual Disabilities (Totsika et al., 2017), Autism (Lang et al., 2010) and ADHD (Daley et al., 2014), it is most parsimonious to assume that they also work in children with epilepsy, until proven otherwise. Therefore, in the absence of epilepsy-specific guidance, Wagner & Smith (2006) suggest that clinicians should use evidence-based interventions with routine outcome measurement (p. 47).\n\nHowever, it is not clear that children with epilepsy are accessing these evidence-based treatments, and in many cases the difficulties remain ‘under-recognised and under-treated in clinical settings’ (Pattanayak & Sagar, 2012, p. 16). For example, Hanssen-Bauer & colleagues (2007) found 77% of 74 children and young people with epilepsy had a probable mental health disorder, but 80% of this group had no contact with psychology or psychiatry, a finding corresponding to other studies (Ettinger et al., 1998; Ott et al., 2003). This finding of an unmet need is not new and indeed warrants further investigation. These studies also demonstrate that a small proportion of children and young people do receive support for their mental health needs but there is little research exploring what this treatment consists of and whether it is compliant with national recommendations for children with identified mental health needs. The primary aim of this study was therefore to identify what psychological treatment young people with epilepsy with mental health needs receive in routine practice. The secondary aim was to establish whether the treatment received was compliant with NICE recommendations for the mental health disorder.\n\n\nMethods\n\nThis cohort study formed part of a larger unpublished study investigating the feasibility of a randomised control trial for treatment of mental health difficulties in children and young people with epilepsy (NIHR Programme Development Grant RP-DG-0614-10003). It received ethical approval from the South East Coast – Surrey Research Ethics Committee (15.LO.1881) and R&D approval from Great Ormond Street Hospital for Children NHS Foundation Trust. We used the STROBE cross sectional checklist when writing our report (von Elm et al., 2018).\n\nParticipants were parents of children and young people aged 3–18 years attending paediatric epilepsy clinics at any of the participating recruitment sites (North East London Foundation Trust; Great Ormond Street Hospital for Children NHS Foundation Trust; Whipps Cross University Hospital; University College London Hospitals NHS Foundation Trust and Whittington Health NHS Trust) between January 2016 and June 2016. The exclusion criteria for this study were minimal and restricted to those with profound intellectual disability.\n\nParticipants were asked to complete the Strengths and Difficulties Questionnaire (SDQ: Goodman, 1997). Those who met threshold for significant emotional or behavioural symptoms were asked to complete the full Development and Well-being Assessment (DAWBA), an online clinical assessment designed to generate psychiatric diagnoses (Goodman et al., 2000). Caseness on the SDQ was defined as the combination of raised symptom score (≥14 out of a maximum of 40) and raised impact score (≥2 out of a maximum of 10; Goodman et al., 2002). Implicit consent was given for SDQ completion (as this is completed as part of routine practice) and full written informed consent was obtained for participants who scored above the threshold for impairing symptoms on the DAWBA.\n\nFollowing DAWBA completion, parents completed a bespoke questionnaire measure containing questions which addressed their experience of accessing support for their child’s mental health difficulties. This ‘Experience of Support’ questionnaire measure was designed specifically for this study by the research team with input from families of young people with epilepsy. The questionnaire included open questions about what treatment they had been offered, the duration of support and when this was offered (Supplementary File 1). This questionnaire was completed electronically by participants. Participants were not offered any specific interventions or referrals for intervention.\n\nOf the 121 participants who reached caseness on the SDQ, 46 parents completed the DAWBA (child age M = 116.15 months, SD = 46.43), 19 (41%) males; of these 29 (63%) met diagnostic criteria for at least one DSM5 disorder. The sample size is based on guidance regarding feasibility studies (Julious, 2005), therefore once 46 participants had completed the DAWBA we closed recruitment.\n\nData was extracted and coded from the Experience of Support questionnaire by an independent clinically trained research assistant. Treatment was coded as useful if participants explicitly stated that they had found the support they had received useful, or indicated benefit, in answer to question 5. Results were analysed using descriptive statistics.\n\n\nResults\n\nOf the 46 participants who completed the DAWBA, 29 (63%) young people met diagnostic criteria for at least one DSM5 disorder. 19 of the 29 children meeting diagnostic criteria (66%) had not received previous support. 16 of the whole sample of 46 (35%) reported that they had experienced previous or current treatment for their child’s difficulties. Of the 16 who did receive treatment, 10 (63%) considered it helpful in addressing their child’s mental health needs. In total, 10 of these 16 children met diagnostic criteria for a mental health disorder according to the DAWBA (63%).\n\nTable 1 presents details of the treatment offered to families. Eight participants reported receiving support within the past year and all aspects of the treatment offered were highly variable. As can be seen from Table 1, whilst some of the interventions may have been compliant with NICE interventions, many were not, for example a child that met diagnostic criteria for Generalised Anxiety Disorder and Autism (according to the DAWBA) received dance therapy.\n\nGAD, general anxiety disorder; CAMHS, Child and Adolescent Mental Health Services; ODD, oppositional defiant disorder; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder.\n\n\nDiscussion\n\nThis study revealed that the majority of young people with mental health needs in epilepsy services were not receiving any intervention. Of those that did receive an intervention, there was a great deal of variability in the mental health support being offered. The findings further strengthen the argument that a large gap in mental health provision exists within this group. Corroborating previous work, a large proportion of the participants with identified mental health needs were not in receipt of adequate support for these problems (Children’s Commissioner, 2016; Ettinger et al., 1998; Hanssen-Bauer et al., 2007; Ott et al., 2003).\n\nThe lack of consistency and inadequacy of treatment for mental health difficulties demonstrated in this study may be owed to the failure to adopt an integrated, collaborative approach to mental and physical healthcare (Naylor et al., 2016). Further, although evidence does exist for the treatment of mental health difficulties in children and young people, mental health clinicians may be reluctant to extrapolate this to children with epilepsy because of questions regarding their utility, efficacy and safety in this group. Some may hold the view that children with epilepsy need different approaches to those without epilepsy and the absence of treatments consistent with NICE guidelines demonstrated in this study may be reflective of such a belief.\n\nTo our current knowledge, this study is the first to formally investigate the routine treatment received for mental health problems within children and young people with epilepsy. However, in terms of the study’s limitations, the sample size is small and limited in representativeness given the clinical sample. Additionally, it was conducted on the basis of parent report, which may be vulnerable to inaccuracies as parents may not correctly remember details of the support they received. Further, the lack of detail regarding the content of the treatment also limits our ability to definitively conclude how compliant the support was with current guidelines. Finally, we recognise that with the absence of a control group, the possibility that this problem may not be specific to children with epilepsy, cannot be ruled out. Instead, this study may serve as another reflection of the problem with regards to Child and Adolescent Mental Health services (CAMHS) access and use of evidence based treatments that exist more generally (Children’s Commissioner, 2016).\n\nIn conclusion, the usual treatment for mental health in children and young people attending paediatric epilepsy clinics is highly variable and inadequate. Providing appropriate, evidence-based treatment is a priority given the enduring impact that poor mental health has on children’s quality of life. Further research should investigate reasons for the lack of treatment following existing guidance for mental health difficulties in children, as well as possible solutions to this. For example, qualitative studies of clinicians’ beliefs about mental health treatment in epilepsy may be beneficial.\n\n\nConsent\n\nWritten informed consent for publication of research based on analysis of anonymised data were obtained from the participants.\n\n\nData availability\n\nNHS England has strict policies on data sharing with which the authors must be compliant. Participants did not provide explicit consent for their data to be publically available. Therefore data may not be made publically available due to ethical restrictions imposed by NHS England. The relevant anonymised data is summarised in Table 1 and may be made available to qualified researchers. Data requests may be sent to the corresponding author of this paper or to ich.psychmedresearch@ucl.ac.uk.",
"appendix": "Grant information\n\nThis study was funded by a National Institute for Health Research Programme Development Grant (Grant Number RP-DG-0616-10007).\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\nThis work was supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.\n\n\nSupplementary material\n\nSupplementary File 1: Experience of Support questionnaire.\n\nClick here to access the data.\n\n\nReferences\n\nBaca CB, Vickrey BG, Caplan R, et al.: Psychiatric and medical comorbidity and quality of life outcomes in childhood-onset epilepsy. Pediatrics. 2011; 128(6): e1532–e1543. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChildren’s Commissioner: Lightening Review: Access to Child and Adolescent Mental Health Services. London, UK: Children’s Commissioner for England. 2016. Reference Source\n\nDaley D, van der Oord S, Ferrin M, et al.: Behavioral interventions in attention-deficit/hyperactivity disorder: a meta-analysis of randomized controlled trials across multiple outcome domains. J Am Acad Child Adolesc Psychiatry. 2014; 53(8): 835–847. PubMed Abstract | Publisher Full Text\n\nDavies S, Heyman I, Goodman R: A population survey of mental health problems in children with epilepsy. Dev Med Child Neurol. 2003; 45(5): 292–295. PubMed Abstract | Publisher Full Text\n\nde Araujo Filho GM, Yacubian EM: Juvenile myoclonic epilepsy: psychiatric comorbidity and impact on outcome. Epilepsy Behav. 2013; 28 Suppl 1: S74–S80. PubMed Abstract | Publisher Full Text\n\nEttinger AB, Weisbrot DM, Nolan EE, et al.: Symptoms of depression and anxiety in pediatric epilepsy patients. Epilepsia. 1998; 39(6): 595–599. PubMed Abstract | Publisher Full Text\n\nGoodman R: The Strengths and Difficulties Questionnaire: a research note. J Child Psychol Psychiatry. 1997; 38(5): 581–586. PubMed Abstract | Publisher Full Text\n\nGoodman R, Ford T, Meltzer H: Mental health problems of children in the community: 18 month follow up. BMJ. 2002; 324(7352): 1496–1497. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodman R, Ford T, Richards H, et al.: The Development and Well-Being Assessment: description and initial validation of an integrated assessment of child and adolescent psychopathology. J Child Psychol Psychiatry. 2000; 41(5): 645–655. PubMed Abstract | Publisher Full Text\n\nHanssen-Bauer K, Heyerdahl S, Eriksson AS: Mental health problems in children and adolescents referred to a national epilepsy center. Epilepsy Behav. 2007; 10(2): 255–262. PubMed Abstract | Publisher Full Text\n\nHirtz D, Thurman DJ, Gwinn-Hardy K, et al.: How common are the \"common\" neurologic disorders? Neurology. 2007; 68(5): 326–337. PubMed Abstract | Publisher Full Text\n\nJones JE: Treating anxiety disorders in children and adolescents with epilepsy: what do we know? Epilepsy Behav. 2014; 39: 137–142. PubMed Abstract | Publisher Full Text\n\nJulious SA: Sample size of 12 per group rule of thumb for a pilot study. Pharm Stat. 2005; 4(4): 287–291. Publisher Full Text\n\nLang R, Regester A, Lauderdale S, et al.: Treatment of anxiety in autism spectrum disorders using cognitive behaviour therapy: A systematic review. Dev Neurorehabil. 2010; 13(1): 53–63. PubMed Abstract | Publisher Full Text\n\nMiller G, Chen E, Cole SW: Health psychology: developing biologically plausible models linking the social world and physical health. Annu Rev Psychol. 2009; 60: 501–524. PubMed Abstract | Publisher Full Text\n\nNaylor C, Das P, Ross S, et al.: Bringing together physical and mental health: a new frontier for integrated care. 2016. Reference Source\n\nNICE: Epilepsies: diagnosis and management (CG137). 2012. Reference Source\n\nNICE: Social anxiety disorder: recognition, assessment and treatment (CG159). 2013. Reference Source\n\nNICE: Antisocial behaviour and conduct disorders in children and young people: recognition and management (CG158). 2013. Reference Source\n\nOtt D, Siddarth P, Gurbani S, et al.: Behavioral disorders in pediatric epilepsy: unmet psychiatric need. Epilepsia. 2003; 44(4): 591–597. PubMed Abstract | Publisher Full Text\n\nPattanayak RD, Sagar R: Psychiatric aspects of childhood epilepsy. Iran J Child Neurol. 2012; 6(2): 9–18. Publisher Full Text\n\nRodenburg R, Stams GJ, Meijer AM, et al.: Psychopathology in children with epilepsy: a meta-analysis. J Pediatr Psychol. 2005; 30(6): 453–468. PubMed Abstract | Publisher Full Text\n\nStevanovic D, Jancic J, Lakic A: The impact of depression and anxiety disorder symptoms on the health-related quality of life of children and adolescents with epilepsy. Epilepsia. 2011; 52(8): e75–e78. PubMed Abstract | Publisher Full Text\n\nTotsika V, Mandair S, Lindsay G: Comparing the Effectiveness of Evidence-Based Parenting Programs on Families of children with and without Special Educational Needs: Short-term and Long-term Gains. Frontiers in Education. 2017; 2(7): ISSN 0190-5848. Publisher Full Text\n\nvon Elm E, Altman DG, Egger M: The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Accessed 20 August 2018. 2018. Reference Source\n\nWagner JL, Smith G: Psychosocial intervention in pediatric epilepsy: a critique of the literature. Epilepsy Behav. 2006; 8(1): 39–49. PubMed Abstract | Publisher Full Text\n\nWeisz JR, Kuppens S, Eckshtain D, et al.: Performance of evidence-based youth psychotherapies compared with usual clinical care: a multilevel meta-analysis. JAMA Psychiatry. 2013; 70(7): 750–761. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "42323",
"date": "31 Jan 2019",
"name": "Charlotte U. Rask",
"expertise": [
"Reviewer Expertise Child and Adolescent Psychiatry",
"functional disorders",
"pediatric liaison psychiatry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study explores the type of psychological treatment that young patients with mental health problems co-morbid to epilepsy receive in routine practice and whether this treatment is in accordance with NICE recommendations. Based on their results, the authors conclude that the treatment offered is inconsistent and inadequate, and that there is a clear need to optimise clinical practice in this area.\n\nThe topic of the paper is interesting and the introduction well-written with clear aims. However, there are some critical points related to the description of used methods and subsequently the interpretation of the results which the authors need to address and clarify:\n\nThe authors state that this study is part of a feasibility study prior to a planned larger randomised control trial for treatment of mental health difficulties in children and young people with epilepsy. However, no clear objectives or details of the design of this feasibility study are provided – the authors only state that they follow the STROBE cross sectional checklist when writing the report. Please provide more information. The participants are recruited from four specialised epilepsy clinics. A bit more clinical information would be helpful, e.g. are the participants all new referrals or is it a mixture of children who have been followed for a long time in the clinics and children who are seen for the first time? This could be interesting in order to understand where the \"gap\" in the health care system is when it comes to identifying and initiating treatment for mental health problems in this patient group. The main measure to identify mental disorders is the DAWBA. DAWBA is a broad diagnostic instrument that involves questionnaires, interviews and rating techniques, and it covers the most common child psychiatric disorders. Versions exist for various age groups (2-4 yrs, 5-17 yrs) and different informants (children from age 11 and parents) who independently of each other answer structured questions, which cover the operationalized diagnostic criteria for a broad spectrum of DSM and ICD diagnoses. Schoolteachers complete a shorter questionnaire. Open-ended questions record the respondent’s own description of problems which makes it possible to perform a supplementary clinical rating in order to increase the diagnostic validity. However, in the current study it is not clear whether age corresponding versions were used. Furthermore, it reads as if no clinical rating of the interviews was performed, which might be a considerable limitation also as only one informant (i.e. one parent) was used to obtain information. Also, it could have strengthened the diagnostic findings if these where supported by information from other sources, e.g. medical records. The authors state that 120 children reached caseness on the SDQ - but not out of how many? A flowchart would have been nice, also showing numbers for attrition in order to evaluate the generalisability of the study findings. Criteria for caseness are based on SDQ scores. Please provide a bit more information on whether these criteria in general are used as standards to define potential cases with significant mental health problems within clinical study samples. Please explain what is meant by the phrase: \"threshold for impairing symptoms on the DAWBA\". Do the authors here refer to whether or not there are positive computer generated diagnoses in the DAWBA interview? The rationale for the chosen sample size is not clear for the purpose of this study. The authors only states that it is based on guidance regarding feasibility studies. N=46 actually seems a bit small to estimate the size of the clinical problem this study wants to elucidate. Another main variable in the study, i.e. provided support for mental health problems, is assessed by a new measure which was not validated before its use. Furthermore, the rating is only performed by one person. This could introduce bias.\nThe mentioned methodological issues mean that the results should be interpreted with caution. They do suggest that there is a problem. However, it may be difficult to estimate the size of the problem based on this study.\nIn sum, the authors need to spend more time critically discussing their findings in relation to the weaknesses of their study design and used 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5218",
"date": "09 Apr 2020",
"name": "Alice Welch",
"role": "Author Response",
"response": "Thank you very much for your extremely helpful comments and suggestions. We have made the following changes and clarifications to the methodology: We have provided further detail on the design and objectives of the feasibility study. We have clarified how the DAWBAs were clinically rated and provided further detail regarding the use of the SDQ within paediatric clinical samples. We have included further recruitment data to highlight the proportion of participants that completed the SDQ. As suggested, we have also added a flowchart to supplement this information (See Figure 1)."
}
]
},
{
"id": "46740",
"date": "07 May 2019",
"name": "Maria Teresa Lax-Pericall",
"expertise": [
"Reviewer Expertise Mental health difficulties in children with epilepsy and brain injury"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study addresses the important question whether children with epilepsy who have mental health difficulties are appropriately identified and managed. It concludes that there is “inconsistency and inadequacy of mental health provision for children and young people with epilepsy”. In the discussion the authors add “This study revealed that the majority of young people with mental health needs in epilepsy services were not receiving any intervention” and that “a large gap in mental health provision exists within this group”. The authors mention the “inadequacy” of some of the interventions received by some of the 16 children. The study was conducted in 6 different paediatric epilepsy clinics, covering a large geographical area over 6 months; therefore should be well placed to address the issue of possible variability and inconsistency in accessing mental health services and treatments provided. However, there are some limitations to the methodology of this study that may influence the conclusions the authors have reached.\n\nMethodology\nSample bias: The study was conducted as part of a feasibility study carried out in paediatric epilepsy clinics. Not all children with epilepsy in the UK are seen in a paediatric epilepsy clinic, there is a proportion of children with epilepsy (in the UK these usually are children with less complex epilepsy) that are seen by general paediatricians. Therefore, it should be clear that the findings apply to a selected population of children with epilepsy (probably children with more complex epilepsy). The authors made this clear in the abstract, but because the frequency of mental health problems is higher in children with epilepsy who have other neurological conditions or changes in the MRI than in children with less complex epilepsy, I think it would be worth marking this point in the discussion.\nThe next issue is that we are told that the SDQ was completed as part of routine practice and that there were 121 SDQs that screened positive. It would be useful to know the total number of patients and the percentage of SDQs completed out of the total number of patients. It could be that not all potential participants completed the SDQ. If the ones that did not complete the SDQ are the parents whose children had mental health problems and are receiving appropriate treatment. I must say that, from a clinician’s perspective this explanation seems unlikely, but the paper is research and therefore this possibility needs to be mentioned as a limitation because it will affect the conclusions reached.\nThe next issue is that of 121 cases with positive screen in the SDQ only 46 completed the DAWBA. It is impossible to know why more than 50% of parents did not complete it. Could it be because at least some of them are receiving treatment and are improving? If these were the case it would certainly affect the conclusions. It may not be possible to know this now, but this should be mentioned in the discussion as a limitation.\n\nDiscussion There is not a section regarding limitations of the study and, in my opinion, there should be, with the conclusions reviewed taking into account the limitations. The authors talk about the inadequacy of some of the interventions received by the 16 children. They take NICE guidelines as “gold standard”. Although, in my opinion, this is a good starting point, there are many disorders for which NICE does not have specific recommendations and, even if there was a guideline for a condition, we do not treat conditions, we treat children. Therefore, one cannot conclude that a treatment given was “inadequate” without knowing much more about that particular child, family and other factors that may influence the decision regarding treatment offered…\nSuggestions\nThe study will benefit from a limitations section in the discussion and reviewing /curtailing their conclusions in the light of these.\n\nIs the work 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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5219",
"date": "09 Apr 2020",
"name": "Alice Welch",
"role": "Author Response",
"response": "Thank you very much for your extremely helpful comments and suggestions. We have made the following changes and clarifications to the methodology and discussion: We have included further recruitment data to highlight the proportion of participants that completed the SDQ. As suggested, we have also added a flowchart to supplement this information (See Figure 1). We have added to our limitation section within the discussion to consider your point re NICE guidelines as the ‘Gold standard’, limitations to the sample and lack of further information re DAWBA completion. We have reviewed our conclusions in light of these."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1907
|
https://f1000research.com/articles/9-250/v1
|
09 Apr 20
|
{
"type": "Opinion Article",
"title": "Cancer cachexia has many symptoms but only one cause: anoxia",
"authors": [
"Tomas Koltai"
],
"abstract": "During nearly 100 years of research on cancer cachexia (CC), science has been reciting the same mantra: it is a multifactorial syndrome. The aim of this paper is to show that the symptoms are many, but they have a single cause: anoxia. CC is a complex and devastating condition that affects a high proportion of advanced cancer patients. Unfortunately, it cannot be reversed by traditional nutritional support and it generally reduces survival time. It is characterized by significant weight loss, mainly from fat deposits and skeletal muscles. The occurrence of cachexia in cancer patients is usually a late phenomenon. The conundrum is why do similar patients with similar tumors, develop cachexia and others do not? Even if cachexia is mainly a metabolic dysfunction, there are other issues involved such as the activation of inflammatory responses and crosstalk between different cell types. The exact mechanism leading to a wasting syndrome is not known, however there are some factors that are surely involved, such as anorexia with lower calorie intake, increased glycolytic flux, gluconeogenesis, increased lipolysis and severe tumor hypoxia. Based on this incomplete knowledge we put together a scheme explaining the molecular mechanisms behind cancer cachexia, and surprisingly, there is one cause that explains all of its characteristics: anoxia. With this different view of CC we propose a treatment based on the physiopathology that leads from anoxia to the symptoms of CC. The fundamentals of this hypothesis are based on the idea that CC is the result of anoxia causing intracellular lactic acidosis. This is a dangerous situation for cell survival which can be solved by activating energy consuming gluconeogenesis. The process is conducted by the hypoxia inducible factor-1α. This hypothesis was built by putting together pieces of evidence produced by authors working on related topics.",
"keywords": [
"Cancer cachexia",
"anoxia",
"intracellular lactic acidosis",
"weight loss",
"nutritional supplements",
"quality of life",
"Cori cycle",
"dichoroacetate",
"emodin",
"pentoxifylline"
],
"content": "Introduction\n\nCancer cachexia (CC), also known as cachexia-anorexia syndrome1, is a consequence of cancer in which patients lose weight with an overall decline in health2. It is a combination of starvation and metabolic disturbances3 that greatly affects quality of life disrupting regular daily activities. In the more advanced states, cancer anorexia-cachexia cannot be modified or improved by increased feeding, stimulating appetite, or nutritional supplements4.\n\nThe cardinal clinical points of this syndrome are5:\n\nProgressive and relentless weight loss (more than 5% of loss compared with normal weight).\n\nLoss of muscle mass.\n\nLoss of fat tissue.\n\nMinimal or no response at all to usual therapies such as nutritional supplementation.\n\nDifficulties in routine daily activities.\n\nMarked fatigue or asthenia.\n\nLoss of appetite.\n\nProgressive deterioration.\n\nReduced cancer treatment tolerance.\n\nReduced length of life. (There is a correlation between weight loss, rate of weight loss and survival.)\n\nAnemia.\n\nThe reduction in food intake alone does not explain the metabolic changes found.\n\nResting energy expenditure (basal metabolic rate) is increased in some cancers but not in others that also produce cachexia.\n\nAssociated with insulin resistance.\n\nAssociated with acute-phase inflammatory reactants.\n\nIn advanced phases of cancer up to 80% of patients exhibit CC6,7. Complex associations between cancer, host, nutrition, psychological, systemic and environmental factors were thoroughly studied as part of the problem. However, a unified and physiopathological explanation is lacking. This led to consideration of CC as a multifactorial consequence of cancer. Based on the thousands of publications and findings on CC, it is the objective of this work to arrange the multiple pieces of evidence published in the world medical literature and to build a comprehensive and unified picture of the syndrome. Understanding how anoxia, rather than hypoxia, is the main culprit of CC will allow for a different approach to treatment on a rational basis. A certain amount of speculation is involved in the proposed solution of the puzzle; however, this speculation has been kept to a minimum. Only laboratory work and clinical trials can ultimately confirm the validity of the hypothesis developed here.\n\n\nKnown mechanisms of cancer cachexia\n\nAs already mentioned, CC is described as the consequence of cancer-induced loss of appetite (reduced food intake-starvation) and metabolic alterations. It is found frequently in advanced tumors and many authors consider it a paraneoplastic syndrome8. Many mechanisms contribute to unleashing cancer cachexia. Some are known, others are not. However, a superficial look may be misleading because the problem is far more complex than it may seem. There are well known factors that play a role, which are summarized below.\n\nAlthough this may seem to be the main cause, it is not. Pharmacologically improving the appetite or increasing food intake does not solve the problem. Usually, it does not stop progressive weight loss. Anorexia is frequent, but there are many patients who lose weight without a manifest loss of appetite. Important contributory factors to anorexia are depression9,10, and cancer treatments themselves, such as chemotherapy and radiotherapy11,12. However, this treatment-related weight loss does not seem directly associated with cancer cachexia.\n\nThese mediators were found increased in cachexia, such as tumor necrosis factor alpha (TNFα)13–15, ZAG (Zinc-α2-glycoprotein also known as lipid mobilizing factor)16,17, interleukin (IL)-118, IL-619, IL-1520, proteolysis inducing factor (PIF)21, myostatin22, and transforming growth factor-β (TGF-β)23, among others. Levels of glucocorticoids are also increased. These mediators seem to be part of the problem but not the cause. For example, TNFα and IL-6 produce loss of appetite by interacting with hypothalamic receptors that regulate food intake24. However, steep elevation of IL-6 is found mainly in very advanced stages of CC25. IL-6 is also involved in an autophagy inducing activity found in serum of patients with CC. Blocking IL-6, this activity disappears26.\n\nA previous study showed that 143 out of 297 (48%) unselected cancer patients exhibited increased resting energy expenditure27. Several authors have confirmed increased energy expenditure as a cause of weight loss in cancer patients28–33. This probably is the consequence of increased uncoupling at the electron transport chain and increased Cori cycle activity. In the liver, the excess lactic acid produced by the tumor can be converted into glucose (Cori cycle) consuming ATP. The Cori cycle is considered an important culprit in cachexia. The Cori cycle (also known as the lactic acid cycle), converts lactate produced by anaerobic glycolysis in muscles to glucose in the liver. In cancer, instead of muscle, the tumor is the provider of lactic acid. While glycolysis produces a positive balance of two molecules of ATPs, the Cori cycle uses up six molecules of ATPs. Each turn of the cycle represents a net loss of four ATP molecules34. The Cori cycle has been held responsible for energy loss in cancer by many authors35–43. An intracellular Cori cycle cannot be ruled out as the main cause of energy loss (Figure 1).\n\nThe figure shows that six ATP molecules are necessary to convert two molecules of lactate into glucose (left panel). On the other hand, the glycolytic pathway (right panel) produces two ATP molecules by degrading glucose to lactate. If two molecules of lactate produced through glycolysis are reconverted into glucose, there is a net loss of four ATP molecules. If this is established as a permanent circuit glucose-lactate-glucose circuit, each turn in the circuit loses four ATP molecules.\n\nInterestingly, the Cori cycle is activated during fasting44 where it contributes to generating glucose. Patients with advanced metastatic cancer show an increased Cori cycle, particularly those patients with high glycolytic flux35,36,45,46.\n\nAccording to our criteria, the Cori cycle by itself, is the main responsible actor in CC, but not the originating cause. Figure 2 and Figure 3 show a proposed mechanism of how the Cori cycle develops in cancer and the energy imbalance it drives. For each molecule of glucose produced through the Cori cycle, six molecules of ATP are used. For each molecule of glucose degraded to lactic acid only two molecules of ATP are produced. Therefore, if a cycle is established in which one molecule of glucose produces two ATP molecules, and the lactic acid thus formed is used to regenerate glucose, four molecules of ATP are lost in each complete cycle.\n\nLeft panel shows condition 1 (slightly hypoxic environment) while the center panel shows condition 2 (extreme hypoxia or anoxia). Right panel shows the effects of anoxia on the energy balance.\n\nThe figure shows how a further turn of the cycle increases glycolytic flux and at the same time increases energy loss.\n\nLoss of adipose tissue due to increased lipolysis47 seems to be activated by protein kinase A48. Hepatic nuclear factor-4 (HNF4) mRNA was downregulated in adipose tissue of patients with CC49. Degradation of triglycerides also has a role in the loss of adipose tissue50 with the intervention of adipose triglyceride lipase51. ZAG (lipid mobilizing factor), which decreases lipids from adipocytes52, also increases the expression of uncoupling proteins in adipose tissue and skeletal muscle and therefore produce potential energy loss53,54. Interestingly, the expression of ZAG is increased with hypoxia and induces insulin resistance55.\n\nLoss of skeletal muscle occurs due to increased proteolysis and decreased protein synthesis.\n\nTumor stage seems to be a predictive factor of cancer cachexia56 and is probably related to tumor mass.\n\nAsp et al.57 found that CC bearing mice had a significantly decreased glucose response to insulin. Rosiglitazone improved insulin sensitivity. Muscle wasting seems to be also related to insulin resistance58. HIF-1α can induce insulin resistance59. Intermittent hypoxia has the same effect60–63.\n\n\nTumor bioenergetics\n\nIf we look at tumor bioenergetics as a highly dynamic process that constantly adapts metabolism to oxygen and nutrients availability, we understand that most cancers have three types of cells according to their glucose metabolic behavior:\n\na) Oxidative when oxygen availability is high (normoxic behavior).\n\nb) A variable mix of glycolytic and oxidative with or without hypoxia. The Warburg effect in this case is the preference for glycolytic rather than oxidative pathway.\n\nc) Fully glycolytic when oxygen is absent (anoxic behavior).\n\nThese three types of metabolic behavior may be present in the same tumor and vary in proportion as the tumor progresses. In very advanced tumors or very bulky ones, anoxic behavior predominates and causes CC.\n\nFigure 3 and Figure 4 represent a theoretical exercise of what would happen with 100 molecules of glucose in two different environmental conditions:\n\nThese causes are showed separately. However, there is a close interaction among them. The only factor that interacts causatively with all of them is anoxia. Anoxia induces the expression and secretion of inflammatory mediators in macrophages. Anoxia also induces insulin resistance and through HIF-1α activates the Cori cycle. This cycle produces higher energy expenditure. Inflammatory mediators induce decreased nutritional intake through loss of appetite and induce skeletal muscle degradation, lipid deposit loss and finally loss of weight. All the roads lead to anoxia as the coordinator of this syndrome.\n\nThis condition is characterized by a pO2 higher than 0.5%. In this situation the oxidative phosphorylation would remain active and 30% or more glucose would be metabolized by mitochondria (Krebs cycle and electron transport chain) to CO2 and H2O. The rest, 70% of glucose would undergo glycolysis to lactic acid. (Warburg effect: preferential glycolytic metabolism in aerobiosis). The 140 molecules of lactate thus formed are expelled to the extracellular space by the monocarboxylate transporters. Oxidative phosphorylation remains operative during the Warburg effect. In a famous debate with Warburg64 in 1956, Sidney Weinhouse stated that Warburg’s concept about tumor cells being unable to oxidize glucose was wrong. Furthermore, Weinhouse showed that glucose can be oxidized to CO2 in cancer at a rate similar to normal cells65. This concept has since been validated by many authors66–73. The amount of oxidative phosphorylation that continues working after the metabolic shift varies considerably among different tumors and duration of hypoxia74.\n\nWhat must be kept in mind is that the Warburg effect is not the shutdown of the oxidative metabolism. It is the predominance of glycolytic metabolism over oxidative metabolism, but oxidative metabolism continues working. Oxidative metabolism may be decreased, equal to or greater than in normal counterparts. The presence of oxygen increases oxidative metabolism in normal and cancer cells; however, this increase is much lower in the latter. Oxidative metabolism is present even in highly glycolytic cells but operating at a lower capacity75.\n\nAs a conclusion, the Warburg effect is not about mitochondrial metabolism impairment (as Warburg thought) but about increased glucose uptake and glycolytic flux as postulated by Weinhouse. There is high metabolic variability among cancer types and also inside a tumor. This means that glycolytic cells may conserve variable degrees of mitochondrial metabolism.\n\nIn this condition, the pO2 has decreased below 0.2%. Mitochondrial activity is practically downregulated by such a low level of oxygen. Therefore, nearly 100% of glucose is degraded to lactic acid, generating 200 molecules of lactate. Such a high lactate load can easily surpass monocarboxylate extruding capacity and a certain amount of lactate would remain inside the cell creating intracellular lactic acidosis that would endanger cancer cell survival. Activation of the Cori cycle comes to solve this situation by reconverting part of the lactate to glucose. This creates a vicious cycle in which the more glucose is degraded, the more the Cori cycle “works”, consuming four ATPs in each turn of the cycle. Each turn of the cycle increases the glycolytic flux by the generation of more glucose. This creates a vicious cycle. Figure 2 and Figure 3.\n\nFigure 2 and Figure 3 are not based on real calculations. They are only used to illustrate reasons why the Cori cycle is activated and how this cycle consumes progressively higher amounts of energy. As we can see the main culprit in this scheme is extreme hypoxia.\n\nThe following concepts were taken into consideration in the construction of Figure 2 and Figure 3.\n\nHypobaric hypoxia has been shown to produce weight loss through diverse mechanisms including loss of appetite and activation of the Cori cycle76. HIF-1α and HIF-2α are strongly increased with ascent above 4,000 meters of altitude77. HIF-1α is a transcriptional activator of phosphoenolpyruvate carboxykinase (PEPCK) which is the rate-limiting enzyme for gluconeogenesis78. Experimental downregulation of HIF-2α decreased gluconeogenesis in hepatoma cells (HepG2) and decreased tumor size79.\n\nSuhara et al.80 have shown that gluconeogenesis (Cori cycle) is a mechanism that defends against lactic acidosis. Why does the cancer cell need the Cori cycle? The need stems from the fact that the monocarboxylate transporter (MCT) system is saturable. The complete or almost complete abrogation of the mitochondrial metabolism plus the increased glycolytic flux represent such a burden that the MCT capacity is surpassed. In muscle, the half-maximal rate of lactate transport is achieved with a lactate concentration between 13 and 40 mM81. If the maximal rate is achieved (about 20 nmol/min per μl of intracellular volume at 25°C)82, the excess would remain inside the cell. Therefore, by transforming lactate into glucose or pyruvate The Cori cycle prevents intracellular lactic acidosis which would induce acidic stress and kill the cell. The velocity of lactate extrusion by MCTs is also dependent on intracellular and extracellular pH60. Decreased intracellular pH increases extrusion velocity, while it is lowered by extracellular acidity59. Anoxic areas of tumors have a very acidic extracellular substance, and this may slow down lactate extrusion.\n\nKoloyianni et al.83 and Ludholm et al.38 found that in normal cells, 60% of glucose generated by gluconeogenesis used lactate as the source molecule. Glutamine and alanine contributed 10% each and glycerol 3%. The rest came from serine, glycine, and threonine.\n\nPhenformin inhibits gluconeogenesis84. This is a paradoxical result in the scheme, because Complex I inhibition decreases oxidative phosphorylation. However, this anti-gluconeogenesis activity of Complex I has been tested in cells that were still performing oxidative phosphorylation. It is possible that under full anaerobiosis Complex I inhibition would have no effect on gluconeogenesis.\n\nA proof of this last concept is that pharmacological inhibition of HIF-1α reduces cancer cachexia85. The authors used emodin and rhein (from Rheum palmatum) to decrease HIF-1α expression. Interestingly, emodin and rhein have been shown many other anti-cancer effects86–89.\n\nMitochondrial diseases that decrease mitochondrial activity can produce lactic acidemia90. The same happens with excessive anaerobic exercise91,92. However, in the case of exercise, even though the ability of MCTs to expel lactate is not exceeded, there is no intracellular lactic acidosis.\n\n\nCan anoxia by itself explain the production of inflammatory and catabolic mediators?\n\nIn 1991, Ghezzi et al.93 showed that anoxia/hypoxia with very low levels of endotoxin was able to increase levels of TNFα, IL-1α, and IL-1β more than twofold. West et al.94 further confirmed these findings and added IL-6 and prostaglandin E2 to the previous list of increased cytokine production by macrophages. Macrophages were activated in an anoxic environment95. IL-8 production is also increased in macrophages under hypoxic conditions96. All these findings were confirmed by many authors97–102. Macrophages resistant to hypoxia modify their phenotype and achieve a high production of inflammatory mediators103. It is highly possible that the inflammatory mediators found in CC are the products of macrophages associated with the tumors that are subjected to the same extreme hypoxic conditions as tumors.\n\n\nCan anoxia by itself explain insulin resistance?\n\nInsulin resistance is frequently found in patients with CC104–107. Yoshikawa et al.107 found that insulin resistance in cancer patients was not caused by malnutrition.\n\nIntermittent hypoxia induces insulin resistance108,109. Under normal conditions insulin is a down-regulator of gluconeogenesis. With the development of insulin resistance this, inhibition is handicapped.\n\nUsually, tumors suffer intermittent hypoxia/anoxia rather than a permanent condition. Growth, invasion and angiogenesis create a very dynamic environment with variable conditions of oxygenation110–112. Furthermore, insulin resistance is a necessary development for Cori cycle activation113, because insulin is the main downregulator of gluconeogenesis114. The following circuit is probably functional in CC:\n\nAnoxia ➔ Insulin resistance ➔ Gluconeogenesis\n\nTNFα is also an inducer of insulin resistance in adipocytes115 and in other tissues116,117. TNFα is a predictor of insulin resistance in pregnancy118. Saghizadeh et al. found that TNFα expression was fourfold higher in the muscle of individuals with insulin resistance compared with healthy normal controls119. Noguchi et al.120 found that TNFα increased expression was associated with insulin resistance in the skeletal muscles of cancer patients. Therefore, another circuit is probably operating in CC:\n\nAnoxia ➔ TNFα ➔ Insulin resistance\n\nIL6 and IL8 also play a role in insulin resistance121.\n\n\nCan anoxia by itself explain lipolysis?\n\nBriançon-Marjollet et al.122 found that endothelin-1 (ET-1) was overexpressed in adipose tissue with intermittent hypoxia and this protein activated lipolysis.\n\n\nAnoxic growth versus hypoxic growth\n\nSince the seminal works by Semenza123–126, it was well established that cells grown in hypoxic medium stabilize HIF-1α, which binds HIF-1β acting as a transcription factor dimer for genes known as hypoxia responsive genes. Therefore, stable HIF-1α expression is a signal of cellular hypoxia (with the exception of those cases where HIF-1α is constitutively activated like Von Hippel Lindau disease). If, instead of hypoxia, the tumor cell is grown under anoxia, something different happens:\n\n1) in the first 3–10 days, HIF-1α is highly expressed;\n\n2) after the 10th day, HIF-1α is not expressed any more127.\n\nIn both cases inflammatory cytokines are increased many folds compared with normoxic cells. The difference between the first ten days and after that is that the cell has become fully anaerobic. The authors stated “Thus, metabolically active HeLa cells respond to the lack of oxygen, in part, by regulating the levels of cytokines produced”. The increase in cytokine production was higher after 10 days of anoxia as compared with 3 days anoxia. This research clearly shows the difference in cytokines expression between short and prolonged anoxia. Figure 4 summarizes the concepts discussed above.\n\n\nUsual treatments of CC\n\nMany treatments have been used for CC. Not one has shown really encouraging results. Most of them address improving appetite and increasing food intake and/or supplementing calories. Other treatments target the intermediary chemokines such as tumor necrosis factor. Why all these failures? All the treatments used so far counter the symptoms and collateral effects of CC. None of them target the main (and unique) cause which is anoxia, or the main mechanism by which anoxia produces its effects, namely gluconeogenesis (Cori cycle). The failed therapies include:\n\nHydrazine sulfate, which has been tested in cachexia treatment but has not yielded any appreciable results128,129. However, some favorable results have been reported130. “Hydrazine sulfate has shown no anticancer activity in randomized clinical trials, and data concerning its effectiveness in treating cancer-related cachexia are inconclusive”131. It has not been approved by the FDA for any medical condition.\n\nSteroids. Different steroids such as medroxyprogesterone132–134, megestrol135–137 have been tested for cachexia treatment. Megestrol have shown beneficial effects limited to appetite, however it does not impact cachexia and is associated with many side effects138.\n\nEicosapentaenoic acid. This is an omega-3 (n-3) polyunsaturated fatty acid (PUFAs). It targets the loss of muscular mass, but does not solve the other effects of CC139,140.\n\nHigh calorie nutritional supplements.\n\nHigh dose progestins. These did lead to some appetite and weight improvement but without major results in the relentless evolution of CC141.\n\nEtanercept and infliximab have been used as anti-TNFα with poor results in CC142.\n\nTocilizumab is an anti-IL6 antibody approved by the FDA for rheumatoid arthritis treatment. It showed benefits in some cases of CC143–145. However, there are no randomized clinical studies confirming these benefits. The number of cases that have been published are scarce.\n\nInsulin for the treatment of insulin resistance. Lundholm et al.146 found insulin as an important palliative treatment for patients with cancer-related weight loss.\n\nRosiglitazone for insulin resistance treatment.\n\nCOX2 inhibitors like celecoxib for decreasing acute phase pro-inflammatory cytokines. A pilot study with celecoxib showed beneficial effects in patients with CC147.\n\nIn the next section we shall propose a treatment scheme based on targeting the physiopathology of CC, rather than the secondary symptoms.\n\n\nDiscussion and hypothesis\n\nFrom a metabolic point of view, there are three types of cells in most tumors:\n\na) Oxidative cells located near blood vessels with adequate or near adequate oxygen and nutritional supply.\n\nb) Aerobic glycolytic cells located in the tumor mass with inadequate oxygen supply but with a functional oxidative phosphorylation that metabolizes part of the glucose to CO2 and H2O; however, these cells preferentially and in major proportion use the glycolytic pathway to lactic acid.\n\nc) Deeply anaerobic cells with near zero supply of oxygen in which only glycolysis to lactic acid is functional. These cells are unable to perform oxidative phosphorylation.\n\nThe proportion of each of these phenotypes in a tumor are variable and dynamic. At an early stage and in small tumors probably oxidative and glycolytic aerobic cells are found. As the tumor continues growing, the severely anaerobic cells, appear. The reason for this is mainly anatomic: they are in completely oxygen deprived areas. Since this last group of cells is incapable of using oxidative phosphorylation it is fully glycolytic and its lactic acid output is higher than in the other two groups.\n\nFully anaerobic cells have three characteristics:\n\na) very high level of HIF-1α expression and activation;\n\nb) high level of intracellular lactic acid which surpasses the extrusion capacity of monocarboxylate transporters;\n\nc) a tendency towards intracellular lactic acidosis.\n\nHIF-1α upregulates PEPCK, activating the Cori cycle. This creates an increased energy imbalance due to a loss of four ATPs for each complete glucose-lactate-glucose cycle. In spite of the energy imbalance thus created, the new scheme rescues the cell from death due to intracellular acidification and restores the intracellular alkalinity needed for adequate proliferation.\n\nWhen the proportion of severely anaerobic cells in a tumor increases, cancer cachexia develops. Usually this is the result of tumors with poor vascular supply and/or large size.\n\nMany pro-cachexia tumors produce proteins and cytokines (whether by themselves or by stimulating other tissues) that have a lipolytic or miolytic effect such as myostatin148. TNFα and interferon-γ produce loss of appetite and consequently decreased food intake149.\n\nWhat is the evidence sustaining the above hypothesis?\n\n1) The lactate shuttle is the best proof of the coexistence of oxidative cells and aerobic glycolytic cells.\n\n2) Frequent findings of necrotic areas in large tumors prove the existence of cells that are extremely anoxic but could not implement the salvage through the Cori cycle.\n\n3) Cancer cachexia frequently appears in the late stages of malignant progression.\n\n4) Cancer cachexia appears progressively, as certain tumors increase in size150.\n\nThe sequence of events leading to CC is shown in Figure 5 and Figure 6.\n\n1a, 2a, 3a, 4a, 5a, 6a,7a, and 8a indicate the relation of anoxia/hypoxia on one side to the chemokines (also called toxohormones by some authors) or direct metabolic effects on the other side.\n\nThe mediator action of cytokines produced by the anoxic cells is related to loss of appetite, loss of muscle and adipose mass. HIF-1α is the transcription factor that activates the Cori cycle as a salvage mechanism from the lactate overload. The center of all these activities is extreme hypoxia/anoxia/intermittent anoxia.\n\nFigure 6 is an integrated view of the relationship between anoxia/deep hypoxia/intermittent hypoxia, on one side and the intermediaries leading to the cardinal symptoms of CC. Also the relationship among these intermediaries has been included in the drawing. This figure shows the essential link between anoxia and CC. The figure is based on the following references: References on Figure 6: 1a)151–155, 1b)156–159; 2a)160–165; 2b)166–169; 3a)170–172; 3b)173,174; 4a)80,175–177; b)178–180; 5a)62,63,181–184; 5b)185,186; 6a)187–191; 6b)192; 6c)193–195; 6d)196–200; 6e)57; 7a)126. IL-6 stimulates the production of endothelin-1 (ET-1)201, not shown in the figure. ET-1 is also a potent vasoconstrictor increasing anoxia202, not shown in the figure; 7b)126,203,204. ET-1 also induces insulin resistance205, not shown in the figure; 8a)206; 8b)207–211; 9)13; 10) Increased glycerol-3-phosphate production, whether from glycolysis or the Cori cycle stimulate adipose tissue loss under hypoxia212; 11) Insulin resistance increases lipolysis213,214.\n\n\nThe role of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)\n\nPGC-1α is a 798-amino-acid transcriptional coactivator considered an important activator of mitochondrial biogenesis215,216. Interestingly, this protein has some characteristics that are important in CC:\n\nPGC-1α is a coactivator for the transcription of other proteins that act in energy metabolism.\n\nIt determines lactate metabolism217.\n\nIt activates mitochondrial fatty acid β oxidation218,219.\n\nPGC-1α induces gluconeogenesis220.\n\nIt activates thermogenic genes, increasing energy expenditure221.\n\nPGC-1α can be recruited by estrogen related receptors222,223.\n\nAnd most importantly, it is increased in hypoxic conditions224 in different tissues including central nervous system225.\n\nPGC-1α, a hypoxia inducible coactivator protein increases thermogenesis and loss of energy and induces mitochondrial lipolysis. PGC-1α shows also other pro-tumoral activities in:\n\nGlioblastoma, where it determines a more aggressive phenotype226.\n\nER-negative breast tumors, in which the level of estrogen related receptors is significantly increased and probably interacting with PGC-1α. Furthermore, the association of PGC-1α with estrogen related receptors positively regulates HIF-2α transcription227.\n\nColorectal cancer, in which hypoxia induced over-expression of PGC-1α regulates tumorigenesis, enhancing cell motility, proliferation, stemness, resistance to chemotherapy, and reducing ROS by antioxidant enzymes activation228.\n\nMelanoma, which when PGC-1α is overexpressed, cells show oxidative metabolism229,230.\n\nOur interpretation of PGC-1α over-expression in the CC is that cells try to activate the mitochondrial oxidative metabolism in a process where anoxia has near shut down this metabolic pathway. Therefore, PGC-1α is a hypoxia modulated protein that is able to control many of the intermediary steps between anoxia/hypoxia and the symptoms of CC.\n\n\nTreatment proposal\n\nIn this paper we argue that anoxia is the main cause of CC. Figure 5 and Figure 6 shows how anoxia achieves all the cardinal symptoms of CC through diverse mechanisms. The figure does not show the intracellular lactic acidosis, because the Cori cycle prevents its onset.\n\nBased on Figure 5 and Figure 6 we must first target anoxia and the Cori cycle. Then we can attack the symptoms. Going against the symptoms alone has failed consistently in the past.\n\nThe scheme proposed here:\n\nAnoxia is difficult to target. Vasodilators such as nitrates and “hemodynamic improvers” such as pentoxifylline may improve circulation in the tumor decreasing anoxia. Flunarizine is a vasodilator with the ability to block voltage gated sodium channels. Nitroglycerin is another vasodilator that has been tested in tumors. A combination of these drugs should enhance tumor oxygenation. Any anti-angiogenic drugs being used, should be discontinued.\n\nHIF-1α, directly upregulated by anoxia, is a targetable transcription factor. Although extensive and intensive research for an inhibitor has been going on for many years, no adequate drug has been developed yet. Emodin is a non-toxic inhibitor of HIF and may produce some benefits231–235 in CC.\n\nIntracellular lactic acidosis-Cori cycle-gluconeogenesis: one of the mechanisms to decrease Cori cycle is to impede excessive lactic acid production, whether in the anaerobic cancer cells or in the liver. In this sense probably the drug of choice should be dichloroacetate (DCA).\n\nImproving appetite: if the previous issues have been medicated and controlled to a certain extent, then improving appetite with megestrol and administering high calorie nutritional supplements makes sense.\n\n\nThe fundamentals for the treatment scheme\n\nDCA is an investigational drug for lactic acidosis, pulmonary hypertension and now for cancer. In some African countries, its use is unofficially accepted for the treatment of lactic acidosis in children with malaria236. It is a small molecule with the following formula HOOC-CH2-Cl2. DCA is an orally available molecule that is almost completely and quickly absorbed at the digestive system237,238. It is metabolized by GSTZ1 (a glutathione transferase isoform)239.\n\nWhen DCA was administered to 16 healthy individuals, in 1 to 50 mg/kg IV infusions, it lowered plasma glucose, lactate and alanine concentrations. Plasma levels linearly followed those of the administered dose up to 30 mg/kg. A dose of 35 mg/kg was considered most effective regarding lactic acid, which fell to 75% below baseline concentrations within 2 hours of the infusion240. Blood glucose was not affected in these healthy individuals but was reduced in diabetic patients through stimulation of peripheral glucose utilization and inhibition of gluconeogenesis. DCA also inhibits lipogenesis and cholesterol synthesis. Daily oral administration of 50 mg/kg DCA to diabetic patients with slightly elevated lactic acid concentration, reduced alanine and lactic acid in plasma241.\n\nThe experimental administration of a single dose of DCA may be misleading upon dose and metabolism, because as Gonzalez-Leon et al.242 have shown in rats with repeated administration of DCA, this drug has a surprising feature: it inhibits its own metabolism. This means that the chronic administration of DCA differs from single doses in its plasma concentration, toxicology and metabolism. Figure 7.\n\nThis is an important issue to be considered for the clinical use of DCA.\n\nIn humans, the maximum plasma concentration achieved with an IV infusion of 10 mg/kg was between 19.9 μg/ml and 24.7 μg/ml with a half life of only 20 minutes. When the infused dose was increased to 20 mg/kg the plasma concentration was between 57.3 and 74.9 μg/ml with a half life of 36 minutes. In dogs and rats the half life was much longer (the half life of a 100 mg/kg dose was 4 hours and 24 hours in dogs and rats respectively243. These marked differences among species cast doubts about the possibility of translating the findings in other mammals to humans.\n\nConfirming these inter-species differences, Maissenbacher et al. have found that dogs present enhanced inhibition of DCA degradation and slower clearance than humans and rats due to increased inhibition of GSTZ1244. The DCA metabolic pathway starts with dehalogenation to monochloroacetate and glyoxylate and then it continues to glycine, and the final products are oxalate and carbon dioxide245. The first dose is cleared from plasma faster than subsequent doses, as Gonzalez-Leon et al. have shown; this is probably due to GSTZ1 inhibition by DCA in subsequent doses. This is similar in all species. The decrease in DCA clearance by multiple doses is not a minor issue, because the initial clearance may be reduced to less than 25% of the initial one in successive doses246.\n\nThe main mechanism of action of DCA is the inhibition of pyruvate dehydrogenase kinase (PDK) and its isoforms. This inhibition increases the flux of pyruvate into the mitochondria, promoting glucose oxidation instead of glycolysis247. PDK inactivates the pyruvate dehydrogenase enzyme complex through phosphorylation. By downregulating the activity of this complex, PDK decreases the oxidation of pyruvate in mitochondria and increases the conversion of pyruvate to lactate in the cytosol.\n\nOther mechanisms of action of DCA in cancer can also be found in the medical literature. Stockwin et al.248 described that cytotoxicity of DCA is only achieved in those cells that suffered mitochondrial DNA mutations that “condemn” them exclusively to the glycolytic pathway. Therefore, DCA has features that make presume it will reduce glycolysis, lactic acid production and gluconeogenesis in anaerobic malignant cells or even cause their death.\n\nWhy DCA?\n\nTNFα and IL-1α are inhibitors of pyruvate dehydrogenase and mitochondrial metabolism249,250. DCA has exactly the contrarian effect.\n\nDCA decreases the expression of TNFα and IL-1β and lactate production in ischemic insults251. It also decreases the expression of IL-6 and Interferon γ252.\n\nWhile TNFα increases fermentative glycolysis, DCA decreases it253.\n\nDCA is an inhibitor of lipolysis254.\n\nDCA decreases all the gluconeogenic precursor molecules255 and thus probably decreasing gluconeogenesis.\n\nDCA seems to reduce insulin resistance256.\n\nIt has been suggested that DCA had the ability to decrease/block the Cori cycle257,258.\n\nDCA showed inhibitory effects on HIF-1α in glioblastoma cells259,260.\n\nDCA synergizes with other chemotherapeutic drugs261–266.\n\nDCA targets mainly cells that cannot use the oxidative metabolism248. This concept is further confirmed by the synergy between DCA and metformin267–274. The fundamentals of this association stem from the fact that metformin inhibits mitochondrial Complex I and reduces oxidative metabolism while DCA inhibits glycolysis. This double-edged approach would target very hypoxic cells where oxidative metabolism is minimal and is even further blocked by metformin.\n\nAccording to the effects discussed above, DCA seems the only drug that targets simultaneously most of the proteins and pathways involved in CC pathogenesis.\n\nPentoxifylline reduces the expression of TNFα in cancer cells. It has been tested in CC in five patients with good results in three of them275. Many other reports have confirmed the inhibitory actions of pentoxifylline on TNFα276–279, not only in cancer, but also in other pathologies280,281. Pentoxifylline has other very important actions in CC: it is a hemorheological agent that increases red blood cell deformability, reduces blood viscosity and decreases platelet aggregation improving microcirculation282. Therefore, a better oxygenation of the tumor is expectable. However, a study by Goldberg et al.283 with pentoxifylline as a stand-alone drug did not show any improvement in patients with CC. There is evidence that pentoxifylline increases tumor oxygenation284,285.\n\nNitroglycerin (NTG) is another drug that deserves consideration. It is a well-known vasodilator and oxygenation improver. In addition to these effects NTG also reduces HIF-1α levels in hypoxic tumors286,287, because it acts as a nitrous oxide donor. Unfortunately, NTG has both pro and anti-tumor effects288. However, used on a short-term basis as an adjunct to pentoxifylline, an important improvement of tumor oxygenation can be expected. For a review of nitroglycerin’s anti-tumoral activity, read Sukhatme et al.289.\n\nThalidomide is a TNFα downregulator290 and has been tested in CC with encouraging perspectives291. It had similar results in wasting syndromes of other origins292,293. Thalidomide has also other anti-cancer effects294, such as anti-angiogenesis and T-cell stimulation295, which will not be considered here because they go beyond the scope of this manuscript. It has been established as part of multiple myeloma treatment protocols. For a review of thalidomide, read Luzzio296.\n\nAs mentioned above, emodin can be used for this purpose. Emodin down-regulates HIF-1α expression297. It also inhibits pro-inflammatory responses298 that play a role in CC. Furthermore, hepatic cancer cells (HepG2) treated with emodin showed a significant decrease of lactate. This decrease is a signal of glycolytic inhibition which has been further confirmed because emodin decreased mRNA levels of hexokinase II (HKII), pyruvate kinase isoform M2 (PKM2), and lactate dehydrogenase-A (LDHA) in a concentration-dependent manner299. Emodin also inhibits TNFα, NF-κB and IL-6, all mediators of CC300. Emodin has many other anti-cancer effects, such as:\n\nsensitization to chemotherapeutics235,301–303;\n\nincreasing ROS production304;\n\npromoting apoptosis;\n\ninhibiting angiogenesis305,306, metastasis307, migration and invasion308:\n\ninducing proteasomal degradation of Her2/neu309;\n\ninhibiting ATP citrate lyase310;\n\nincreasing expression of insulin-like growth factor binding protein-1311;\n\nreverting cisplatin resistance312;\n\nblocking STAT 3 activation313, among others.\n\nFor a review on emodin pharmacology see Dong et al.314.\n\nThe treatment proposed here includes the association of pentoxifylline, emodin and DCA as an added scheme to classical treatments such as high calorie nutritional supplements and anabolics like megestrol (Figure 8). Adding these drugs to the conventional treatment would decrease anoxia, HIF-1α expression, and the glycolytic pathway restoring oxidative phosphorylation and reducing TNFα expression. This type of treatments targets the etiology of CC rather than the symptoms.\n\nPEPCK is the rate-limiting enzyme for gluconeogenesis. Therefore, its inhibition should block the Cori cycle. Many drugs have been identified with the ability to inhibit PEPCK, such as metformin315, troglitazone316, berberine317, among others. None of these drugs have been tested in CC. Berberine should be considered a particularly interesting drug because it inhibits PEPCK but also downregulates HIF-1α318. Berberine also has many other anti-cancer effects319, such as down-regulation of COX2320, increased apoptosis in cancer cells without affecting the normal ones321,322, reduced migration and invasion, among others. For a review of other anti-cancer effects of berberine, read Kaboli et al.323\n\n3-bromopyruvate (3BP) is a protein alkylating agent that has shown many anti-cancer effects. We included it in this list of possible drugs for treating CC because it is a potent inhibitor of aerobic glycolysis324. Inhibition of glycolysis should decrease lactate production, thus decreasing the Cori cycle. The exact mechanism of action is not fully known, but there is some evidence pointing to inhibition of glycolytic enzymes325. It has important cytotoxic effects on highly glycolytic tumor cells326.\n\nTocilizumab (a humanized anti-IL6 receptor antibody)327 may produce some benefits328,329. It requires further testing. The association of tocilizumab with gemcitabine for the treatment of advanced pancreatic cancer failed to show clear clinical benefit in a phaseI/II clinical Trial330.\n\nInsulin, besides its known actions (inhibitor of gluconeogenesis), exerts inhibitory activity on PGC1α expression331.\n\nAnamorelin is a small molecule ghrelin receptor agonist that has shown favorable effects on appetite, food intake and weight gain in patients with CC332–334. However, its approval was rejected twice by the European Medicines Agency335.\n\n\nAn alternative hypothesis: the browning of adipose tissue\n\nPetruzzelli et al.336 reported a completely different physiopathological road leading from cancer to cachexia: the browning of white adipose tissue. They maintained that a phenotypic switch from white adipose tissue to brown adipose tissue metabolism was the main culprit of CC. The main characteristic of browning would be the increased expression of uncoupling protein-1 in white adipose tissue, with consequent high energy expenditure. They also found that inflammatory intermediaries (mainly IL-6) were the cause of the browning process and proposed the anti-inflammatory sulindac for CC treatment. Considering that the findings of Petruzzelli et al. are correct, one question remains unanswered: why do advanced tumors produce significant inflammatory mediators? And this takes as back to the anoxia problem: it is anoxia that induces the production of inflammatory mediators.\n\nThe steps between anoxia and CC can be those proposed by Petruzzelli et al. (browning of white adipose tissue) or those hypothesized in this paper (increased Cori cycle). The increased Cori cycle can occur in the tumor, in the liver or in both. However, the primum movens remains anoxia. Treating the intermediate steps (TNFα, IL-6 or other chemokines), or the symptoms (loss of weight, lipolysis, muscle loss) are valid approaches. However, the only significant result would be achieved by simultaneously targeting fermentative glycolysis and anoxia alongside to the other treatments.\n\nA unified explanation of the causes of CC has not been achieved yet337. Therefore, anoxia as the unifying cause behind CC deserves more research.\n\n\nConclusions\n\nA unitary explanation of the cause of CC is presented here. The main culprit of this wasting syndrome is anoxia. The molecular mechanisms leading from anoxia to the full blown syndrome are also presented. A therapeutic approach, based on this hypothesis is proposed.\n\nAnoxia in large areas of the tumor mass is the main cause of CC. This occurs through a sequence of events where oxidative phosphorylation is almost totally shut down leading to full glycolytic behavior (100% of the glucose is degraded through fermentation and none through oxidation). Vascular supply and cell metabolism are highly heterogeneous throughout the tumor. Anoxic anaerobic metabolism is also present in parts of the tumoral mass. When an important portion of the tumor is “pushed” to fully anaerobic metabolism by lack of oxygen, CC develops. Even the most recent publications on CC miss the central issue: anoxia. Therefore, there is no place for anti-anoxic treatments in the therapeutic protocols being used routinely. Anoxia produces such a high level of intracellular lactate that it surpasses monocarboxylate transporters extruder capacities. Thus, the Cori cycle is triggered to prevent intracellular lactic acidosis creating an energetic imbalance due to the cycle’s high energy requirements. Increased inflammatory mediators, that cause many of the symptoms of CC, are not produced by the tumor itself but by hypoxia resistant macrophages associated to the malignant stroma.\n\nAll the treatments employed up to now have failed because they addressed the symptoms of CC instead of the causes. Here we propose targeting anoxia, HIF-1α, and the glycolytic pathway as the logical treatment for CC using a combination of drugs such as pentoxifylline, dichloroacetate, metformin and emodin associated with anabolic steroids and nutritional supplements. The drugs can be changed for others with a similar effect. What is important is to center the treatment on tumoral anoxia, the glycolytic pathway and TNF. It is probably useless to address only one of these issues or expect real improvements with nutritional supplements and appetite improvers. The usually late onset of CC in a prolonged disease and the frequent therapeutic failures have paved the way for a nihilistic attitude that has prevailed up to the present. Targeting the strongly anaerobic cells in the tumor will not only improve CC but at the same time slow down the disease and eventually prolong survival. DCA and the association of DCA with metformin, vasodilators, and HIF-1α inhibitors deserve well planed experimental and clinical research for CC’s therapy.\n\n\nData availability\n\nNo data are associated with this article.",
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PubMed Abstract | Publisher Full Text\n\nVoltan R, Rimondi E, Melloni E, et al.: Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1. Oncotarget. 2016; 7(14): 18965–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaugrud AB, Zhuang Y, Coppock JD, et al.: Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells. Breast Cancer Res Treat. 2014; 147(3): 539–550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKiliccioglu I, Konac E, Albayrak G, et al.: Combination of Metformin and Dichloroacetate Inhibits Proliferation and Induce Intrinsic Pathway of Apoptosis in PC-3 Human Prostate Cancer Cells. Gazi Medical Journal. 2015; 26(4A). Publisher Full Text\n\nDezube BJ, Sherman ML, Fridovich-Keil JL, et al.: Down-regulation of tumor necrosis factor expression by pentoxifylline in cancer patients: a pilot study. 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J Clin Oncol. 1995; 13(11): 2856–2859. PubMed Abstract | Publisher Full Text\n\nZywietz F, Böhm L, Sagowski C, et al.: Pentoxifylline enhances tumor oxygenation and radiosensitivity in rat rhabdomyosarcomas during continuous hyperfractionated irradiation. Strahlenther Onkol. 2004; 180(5): 306–314. PubMed Abstract | Publisher Full Text\n\nLee I, Boucher Y, Demhartner TJ, et al.: Changes in tumour blood flow, oxygenation and interstitial fluid pressure induced by pentoxifylline. Br J Cancer. 1994; 69(3): 492–496. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMateo J, García-Lecea M, Cadenas S, et al.: Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways. Biochem J. 2003; 376(Pt 2): 537–544. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSogawa K, Numayama-Tsuruta K, Ema M, et al.: Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuzzio FA: Thalidomide and Analogues. In Imides. Elsevier. 2019; 367–429. Publisher Full Text\n\nMa F, Hu L, Yu M, et al.: Emodin Decreases Hepatic Hypoxia-Inducible Factor-1 α by Inhibiting its Biosynthesis. Am J Chin Med. 2016; 44(5): 997–1008. PubMed Abstract | Publisher Full Text\n\nHa MK, Song YH, Jeong SJ, et al.: Emodin inhibits proinflammatory responses and inactivates histone deacetylase 1 in hypoxic rheumatoid synoviocytes. Biol Pharm Bull. 2011; 34(9): 1432–1437. PubMed Abstract | Publisher Full Text\n\nXing YX, Li MH, Tao L, et al.: Anti-Cancer Effects of Emodin on HepG2 Cells as Revealed by 1H NMR Based Metabolic Profiling. J Proteome Res. 2018; 17(5): 1943–1952. PubMed Abstract | Publisher Full Text\n\nLu Y, Jeong YT, Li X, et al.: Emodin Isolated from Polygoni cuspidati Radix Inhibits TNF-α and IL-6 Release by Blockading NF-κB and MAP Kinase Pathways in Mast Cells Stimulated with PMA Plus A23187. Biomol Ther (Seoul). 2013; 21(6): 435–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta SC, Rai V: Role of Emodin in Chemosensitization of Cancer. In Role of Nutraceuticals in Cancer Chemosensitization. Academic Press. 2018; 241–257. Publisher Full Text\n\nChen S, Zhang Z, Zhang J: Emodin enhances antitumor effect of paclitaxel on human non-small-cell lung cancer cells in vitro and in vivo. Drug Des Devel Ther. 2019; 13: 1145–1153. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKo JC, Su YJ, Lin ST, et al.: Emodin enhances cisplatin-induced cytotoxicity via down-regulation of ERCC1 and inactivation of ERK1/2. Lung Cancer. 2010; 69(2): 155–164. PubMed Abstract | Publisher Full Text\n\nWang J, Yi J: Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther. 2008; 7(12): 1875–1884. PubMed Abstract | Publisher Full Text\n\nLin SZ, Wei WT, Chen H, et al.: Antitumor activity of emodin against pancreatic cancer depends on its dual role: promotion of apoptosis and suppression of angiogenesis. PLoS One. 2012; 7(8): e42146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaneshiro T, Morioka T, Inamine M, et al.: Anthraquinone derivative emodin inhibits tumor-associated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation. Eur J Pharmacol. 2006; 553(1–3): 46–53. PubMed Abstract | Publisher Full Text\n\nMa J, Lu H, Wang S, et al.: The anthraquinone derivative Emodin inhibits angiogenesis and metastasis through downregulating Runx2 activity in breast cancer. Int J Oncol. 2015; 46(4): 1619–1628. PubMed Abstract | Publisher Full Text\n\nZheng Q, Xu Y, Lu J, et al.: Emodin inhibits migration and invasion of human endometrial stromal cells by facilitating the mesenchymal–epithelial transition through targeting ILK. Reprod Sci. 2016; 23(11): 1526–1535. PubMed Abstract | Publisher Full Text\n\nYan YY, Zheng LS, Zhang X, et al.: Blockade of Her2/neu Binding to Hsp90 by Emodin Azide Methyl Anthraquinone Derivative Induces Proteasomal Degradation of Her2/neu. Mol Pharm. 2011; 8(5): 1687–1697. PubMed Abstract | Publisher Full Text\n\nKoerner SK, Hanai JI, Bai S, et al.: Design and synthesis of emodin derivatives as novel inhibitors of ATP-citrate lyase. Eur J Med Chem. 2017; 126: 920–928. PubMed Abstract | Publisher Full Text\n\nTang Q, Wu J, Zheng F, et al.: Emodin increases expression of insulin-like growth factor binding protein 1 through activation of MEK/ERK/AMPKα and interaction of PPARγ and Sp1 in lung cancer. Cell Physiol Biochem. 2017; 41(1): 339–357. PubMed Abstract | Publisher Full Text\n\nChun-ming QI, Hua-xin HO, Dao-hai CH, et al.: The Reversal Effect of Emodin on Cisplatin Resistance in Ovarian Cancer Cells and the Expression of Resistance-associated Genes. Natural Product Research & Development. 2011; 23(4).\n\nSubramaniam A, Shanmugam MK, Ong TH, et al.: Emodin inhibits growth and induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3. Br J Pharmacol. 2013; 170(4): 807–821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDong X, Zeng Y, Liu Y, et al.: Aloe-emodin: A review of its pharmacology, toxicity, and pharmacokinetics. Phytother Res. 2020; 34(2): 270–281. PubMed Abstract | Publisher Full Text\n\nYuan L, Ziegler R, Hamann A: Inhibition of phosphoenolpyruvate carboxykinase gene expression by metformin in cultured hepatocytes. Chin Med J (Engl). 2002; 115(12): 1843–1848. PubMed Abstract\n\nDavies GF, Khandelwal RL, Wu L, et al.: Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by troglitazone: a peroxisome proliferator-activated receptor-gamma (PPARgamma)-independent, antioxidant-related mechanism. Biochem Pharmacol. 2001; 62(8): 1071–1079. 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PubMed Abstract | Publisher Full Text\n\nMeeran SM, Katiyar S, Katiyar SK: Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation. Toxicol Appl Pharmacol. 2008; 229(1): 33–43. PubMed Abstract | Publisher Full Text\n\nPatil JB, Kim J, Jayaprakasha GK: Berberine Induces Apoptosis in Breast Cancer Cells (MCF-7) Through Mitochondrial-Dependent Pathway. Eur J Pharmacol. 2010; 645(1–3): 70–78. PubMed Abstract | Publisher Full Text\n\nKaboli PJ, Rahmat A, Ismail P, et al.: Targets and Mechanisms of Berberine, a Natural Drug With Potential to Treat Cancer With Special Focus on Breast Cancer. Eur J Pharmacol. 2014; 740: 584–595. PubMed Abstract | Publisher Full Text\n\nFan T, Sun G, Sun X, et al.: Tumor Energy Metabolism and Potential of 3-Bromopyruvate as an Inhibitor of Aerobic Glycolysis: Implications in Tumor Treatment. Cancers (Basel). 2019; 11(3): pii: E317. 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}
|
[
{
"id": "70277",
"date": "09 Sep 2020",
"name": "Marília Seelaender",
"expertise": [
"Reviewer Expertise cancer cachexia"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author intends to explain the symptoms of cachexia by proposing hypoxia as the main driving mechanism. The idea is interesting, but many aspects deserve a more profound view than that presented.\nMajor:\n\nThis is an opinion paper, and this should be very clear in the abstract and introduction, and title (maybe a question mark should be added?). The author claims to have gathered information on all aspects of cachexia in the introduction, but provides no a) methodology for the retrieval and choice of the employed references/studies, b) data, year, findings and type of the study; c) inclusion/exclusion criteria of findings from studies considered. The manuscript cannot be published as it is.\n\nMany relevant aspects of the syndrome are disregarded and may compromise the idea that hypoxia is the driving force of cachexia: for instance, the CNS plays a major role, not only controlling appetite and body composition in cachexia, but also suffers inflammation. Nevertheless, oxygen control to the brain is finely and precisely regulated. How does the author reconcile these data with the affirmed in the manuscript?\n\nReferences are mostly very old, even from textbooks. Recent literature is seldom cited.Therefore a) first be clear that this is a theoretical proposal/opinion, based on biochemistry; b) please consult recent literature on central and peripheral control of cachexia, on the different pattern of immune infiltration and secretion of tumors associated or not with cachexia; and on adipose and muscle.\n\nThe major problem is that the author does not provide whatsoever any evidence on experimental studies in animals, nor patients which could support his hypothesis. Please provide results on hypoxia, blood perfusion and hypoxia markers from the literature;\n\nEnergy expenditure may not be altered in cancer. Although the Cori cycle may be at higher performance (and there is literature available showing that in the liver of animal models, for example - not mentioned in the text), it is nowadays clear that energy demand does not drive cachexia (think of the growing foetus, for instance).\n\nIf hypoxia is causing cachexia-associated lypolisis in adipose tissue, why do obese patients not loose lipids, since their tissue is highly hypoxic?\n\nIf major hypoxia occurs, why do we not see tissue necrosis in cachexia?\n\nThe author blames tumor metabolism/lactate production, yet tumors are composed by many different cells. Do infiltrating immune cells respond to hypoxia? How?\n\nWhy do patients not present muscle cramps, but rather myosteatosis and loss of function? Actually, the mitochondrial carnitinepalmitoyltransferase system activity in augmented in patients with cachexia.\n\nMinor:\nItems in introduction should include; systemic inflammation; muscle quality loss; muscle lipid infiltration.\n\nFig 1: 2 pyruvates/lactates is not the proper form to refer to the molecules.\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? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No",
"responses": []
},
{
"id": "68581",
"date": "09 Sep 2020",
"name": "Barry Laird",
"expertise": [
"Reviewer Expertise Cancer cachexia"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to review this hypothesis generating narrative by Koltai. This is a well written piece of work which aims to present anoxia as the central genesis of cancer cachexia. Literature is critiqued and formulated to support the author's hypothesis. Koltai challenges current scientific thinking behind cachexia arguing that the multimodal approach to treatment has been unsuccessful for decades. The manuscript could highlight that true multimodal trials have been lacking to date and the futility of these can only be supported once robust data has been published. Further it would be interesting to get the author's thoughts as to whether targeting anoxia in combination with multimodal therapies would be a viable therapy.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-250
|
https://f1000research.com/articles/8-1775/v1
|
18 Oct 19
|
{
"type": "Method Article",
"title": "Prediction of cell position using single-cell transcriptomic data: an iterative procedure",
"authors": [
"Andrés M. Alonso",
"Alejandra Carrea",
"Luis Diambra",
"Andrés M. Alonso",
"Alejandra Carrea"
],
"abstract": "Single-cell sequencing reveals cellular heterogeneity but not cell localization. However, by combining single-cell transcriptomic data with a reference atlas of a small set of genes, it would be possible to predict the position of individual cells and reconstruct the spatial expression profile of thousands of genes reported in the single-cell study. To develop new algorithms for this purpose, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) consortium organized a crowd-sourced competition known as DREAM Single Cell Transcriptomics Challenge (SCTC). In the spirit of this framework, we describe here the proposed procedures for adequate reference genes selection, and an iterative procedure to predict spatial expression profile of other genes.",
"keywords": [
"Single-Cell RNA sequencing",
"Drosophila Embryo",
"Gene expression Patterns",
"DREAM Challenge"
],
"content": "Introduction\n\nMulticellular organisms show throughout their development a crescent cellular heterogeneity, distributed and organized in different organs and tissues. This spatial heterogeneity has been explored using different techniques, such as immunohistochemistry and single-molecule fluorescence in situ hybridization (FISH)1. These approaches allow quantification of gene expression in many cells but, unfortunately, these techniques can currently only be assayed to a small number of genes. The selection of these genes introduce a bias that limits the power of these studies. With the advent of emergent methods in genomics, it has become possible to assess the transcriptomic profile of complex tissues with unprecedented resolution, thereby allowing insights into complex processes such as: differentiation trajectories, cell fate decisions, and spatial relationships. In this sense, high-throughput single-cell RNA-seq (sc-RNA-seq) is becoming an established experimental technique2. The protocol of this technique includes the initial step of sample collection, during which solid tissue dissociation results in single cells. Separating cells from their native context results in the loss of spatial information. However, investigating the molecular composition of individual cells in the context of spatial location is important, especially when studying primary cancer cells3. However, some progress has been made to overcome limitations of spatial information loss associated to this technique. Computational methods, based on Principal Component Analysis, are able to partially recover the spatial structure of gene expression patterns4. More recently several computational techniques, coupled to in situ RNA patterns facilitate this reconstruction with better resolution5–7.\n\nIn order to catalyze research on computational methods for the spatial reconstruction of single-cell gene expression data, a crowd-sourced competition was designed by the DREAM Consortium in collaboration with Nikos Karaiskos and Nikolaus Rajewsky from Max Delbruck Institute. Using sc-RNA-seq data from Rajewsky Lab, published in 7, and the expression patterns of driver genes as an expression reference atlas, three main subchallenges were designed. The particular aim was to predict the position of 1297 cells in the 3039 Drosophila melanogaster embryonic locations, or bins, for one half of an embryo in stage 6 (pre-gastrulation), based on scR-NAseq data. The prediction of the 1297 cell positions must be done using a limited number of genes selected from a pool of 84 expression patterns used as a reference atlas. In subchallenge 1, the prediction must be performed using 60 driver genes out of 84 genes; subchallenge 2, using a subset of any expression patterns from 40 genes out of the 84; subchallenge 3, using a subset of any expression patterns from only 20 driver genes. The selection of the subset of genes used for the prediction poses an additional and interesting problem.\n\n\nMethods\n\nExpression patterns used as a reference atlas correspond to 84 driver genes obtained from in situ hybridization experiments; the data correspond to The Berkeley Drosophila Transcription Network Project (BDTNP)8. This gene expression data set is listed in the file bdtnp.csv at DVEX server. One half of the Drosophila embryo has 3039 cells locations, each location is specified by three coordinates (x , y and z) (geometry.txt at DVEX). Thus, the reference database consists of an expression matrix of 84 genes (columns) quantified across the 3039 embryonic locations (rows). These data were binarized following7, sorted in the same order of cell location, and listed in an additional file (binarized_bdtnp.csv at DVEX server). The single-cell RNA sequencing data is provided as a matrix with 8924 genes as rows and 1297 cells as columns. These data are divided by the total number of counts for that cell, in this step a pseudocount is added. The normalized values are obtained by taking the logarithm of the total counts. The normalized values are also binarized, i.e., a given gene is ON (OFF) if the normalized values are above (below) of a quantile value. Based on a distance minimization criterion, the quantile value was chosen as 0.23. The short sequences for each of the 1297 cells in the raw and normalized data are the barcodes of individual cells. Both normalized, as well as binarized, data were provided by the DREAM Challenge.\n\nIn order to select the gene sets to be used in each subchallenge, we take into account two criteria:\n\n(i) Genes that have complementary expression patterns across the single-cell population.\n\n(ii) Genes with expression levels broadly distributed across the single-cell population.\n\nTo accomplish these criteria, we first perform an agglomerative clustering procedure over the expression matrix comprising the 84 genes (the same genes as the available in the in situ expression data) over the 1297 cells. We cluster genes with similar expression profiles across the cells, by means of using the Euclidean distance over the normalized gene expression levels, and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) as a linkage method. Then, we cut the dendogram tree into 20, 40, or 60 groups depending on the subchallenge. This step allows us to identify genes, or cluster of genes, with complementary expression patterns; however, we need to select only one gene per cluster. This selection is performed based on the criterion of the broadest distribution. To this end, for each gene within a given cluster we compute the frequency distribution pi, where pi denotes the frequency of occurrence of expression levels within the bin i. Here, we set the bin size equal to 0.125. After that, we compute the associated entropy S=∑iNpi In pi. Then we select the gene with the greatest entropy in each cluster, i.e., the gene within the cluster with the broadest expression distribution across the single-cell population. This selection procedure is performed with the R script named preprocessing.r, which uses the function selgen.R, both available at Zenodo (see Data availability). To assess this method for the gene selection, we compare the prediction performance obtained with the set of 20 genes selected in this way with the results obtained with different sets of genes sampled at random. For comparison we consider the Mathews correlation coefficient (MCC) between the 1297 cells and the 3039 bins, the ten better scored bins are selected as putative position for each cell. As the true positions of the cells is not available, we take the bin with the highest MCC, obtained with the set that include all 84 genes, as the bin associated with the true position. Thus, we count cells with ten best scores containing the true position as cells whose positions are well predicted. The percentage of the well-predicted positions will be our measure of the performance. Figure 1 depicts the histogram of percentage of cells with well-predicted positions, obtained with 200 sets of 20 randomly selected genes. In all cases this percentage is quite lower than that obtained with 20 genes selected as indicated above, which is 33.46%.\n\nThe performance obtained with the set of 20 genes selected by the proposed method is indicated with a black arrow.\n\nWe use this procedure to select an additional set of 100 genes from the 8924 genes measured by single-cell technique, but excluding the genes from the 84 reference gene set. These 100 genes will be used in further steps during the iterative procedure, and will be denoted as the out group set hereafter. The 20, 40 and 60 selected genes used for each cell location prediction task were listed in Table S1 (see Extended data); we also include the out group set.\n\nIn order to predict the position of a given single cell, we use a score approach based on two similarity measures between the sc-RNA-seq data, and the reference atlas. One of these measures is the Matthews correlation coefficient (MCC) computed between the binarized expression profiles, as proposed in 7. The MCC will be used in the initial step to assign putative bin positions for each single cell and then to predict the spatial expression profile of the outgroup genes. The other measure is the overlap between the normalized expression vector of single cells, and the projected vector corresponding to the predicted spatial expression profile. This vectorial space corresponds to the one spanned by the outgroup genes only. The overlap is defined by: cos(θ)=u⋅vp‖u‖‖vp‖, where u is the profile vector of the single cell and vp is the vector obtained by projecting the profile vector of the predicted profile on the subspace spanned by the non-null components of the profile vector u, as illustrated in Figure 2. The scoring functions are performed by the R script named functions.r, available at Zenodo (see Data availability).\n\n\nResults\n\nThe proposed procedure is schematically illustrated in Figure 3. In the first step we select the set of N genes from the 84 driver genes to be used in the prediction using the method described in Selection of the gene sets section. We also select an additional 100 genes (outgroup genes) from all genes measured in the sc-RNAseq experiment, but excluding the driver genes. The name of the genes used are listed in Extended data: Table S1. Then, using the binarized expression data of the selected genes we compute the MCC (measure 1) for each binarized single-cells vector against the 3039 binarized vectors associated with each positional bin of the reference atlas (BDTNP). By means of the MCC-based score we predict the single cell positions and build the putative expression patterns of the outgroup set of genes. In this sense, the expression level of gen g at the bin position i is given by the weighted average of the normalized gene expression across N putative positions corresponding to that bin, being the weight proportional to the associated MCC. Mathematically, eig=∑j*cijejg, where ci j are the MCC-based scores of the single cell j against position i and eg j are the expression levels of gene g recorded in the individual cells j. The asterisk in the summation indicates that the first better scored N cells are included. The predicted expression patterns computed in this manner are used to compute the overlap (measure 2) with the corresponding expression level of each one of the 1297 single cells. Finally, using the measure 1 and measure 2 we compute a composed score S, defined as S = w1*c + w2*o, where c is MCC-based score, o is overlap-based score and w1 and w2 are the respective weights. The score S is used to predict positions and improve the predicted expression patterns of the outgroup set of genes in each iteration. The last two steps are repeated (2 or 3 times), as indicated in Figure 3 by dashed arrows.\n\nThe above scheme was applied to the Sub-challenges with 20, 40 and 60 genes using different values of the weights. In the first example we apply the procedure to the Sub-challenge 3 and using the 20 genes we compute the MCC for every cell-bin combination. The first iteration of this scoring procedure leads to a performance of 33.5% in assigning the putative positions to each single cell. By means of using the 20 highest coefficients, we predict the expression patterns of the outgroup set of genes. Then, we compute a scoring measure composed of two terms: the MCC computed in the first step (with a weight w1 = 0.7), and the previously-defined overlap between the expression vector of each single cell and the projected expression vector of the reference atlas, being both vectors composed of the 100 outgroup genes (with a weight w2 = 0.3). The score combining both measures is then used to predict the positions of each single cell, which leads to a performance of 36% in the second iteration and 38% in the third iteration. Further iteration steps does not produce any additional improvement. Figure 4A depicts the performance evolution of the procedure using this gene set.\n\nTo select the set of 60 genes to be used in subchallenge 1, from the 84 genes available in the reference atlas, we perform the above mentioned agglomerative clustering procedure. Then, the 60 genes with the greatest entropy within each cluster are selected. The names of the resulting genes were listed in the first column of Extended data: Table S1. As a first step, we compute the MCC for each binarized single-cell vector, and the corresponding 3039 binarized vectors associated with each positional bin of the reference atlas. By means of using the 20 highest MCC for each cell (N = 20), we compute the putative expression patterns of the outgroup set of genes. In this case the used scoring measure was composed by MCC with a weight of 0.90, and the overlap, with a weight of 0.10, of the single-cell expression profiles and the 3039 positions of the predicted expression patterns obtained in the previous step. After two iterations the performance obtained was 95.4%, Figure 4B shows the predicted expression pattern of the ftz gene obtained using the set of 60 genes. The same procedure was used to predict the positions of single cells by considering a set of 40 genes. Again, these genes were selected as described in Methods. The names of the resulting genes were listed in the second column of Extended data: Table S1. In this case the performance obtained reaches 71.4%.\n\nThe expression level of each nuclei is given in white-red scale. Gray nuclei correspond to positional bins without prediction.\n\n\nDiscussion\n\nWe present three innovations that could represent improvements in regard to the original proposal7. One of these innovations is the method for selecting the set of genes to be used as reference in the cell positions prediction task. This set of genes is a good starting point in the presented strategy for position prediction, although we have not explored this method in depth. For example, the Jaccard distance could be used in the clustering procedure instead of the Euclidean distance. We noticed that MCC can overestimate false negatives due to the fact that sc-RNA-seq are not able to record expression of many genes. This results in profiles with many zeros, even in cases of moderate expression levels. For that reason, our second proposed innovation is an alternative way to make the comparison between profiles, as we used in subchallenge 3. Last but not least, the third innovation is the iterative procedure which improves the performance of any of the alternative strategies presented here. In addition, we noticed that the iterative procedure does not necessarily converge to the correct solution, maybe due to error propagation on the predicted patterns.\n\n\nData availability\n\nChallenge documentation, including the detailed description of the Challenge design, overall results, scoring scripts, can be found at: http://www.synapse.org/#!Synapse:syn15665609/wiki. Data for this Challenge can be downloaded from http://shiny.mdc-berlin.de/DVEX/.\n\nZenodo: Prediction of cell position using single-cell transcriptomic data: an iterative procedure, >https://doi.org/10.5281/zenodo.34700619.\n\nThis project contains code and documentation underlying the methods.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nZenodo: Prediction of cell position using single-cell transcriptomic data: an iterative procedure, https://doi. org/10.5281/zenodo.34700619.\n\nThis project contains the following extended data:\n\nTable S1: Selected genes: first, second and third columns list the name of genes used in the Subchallenges 1, 2 and 3, respectively. The last column lists the names of the outgroup genes.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe are grateful to the DREAM SCTC for curation of the challenge and the evaluation of the models.\n\n\nReferences\n\nBattich N, Stoeger T, Pelkmans L: Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat Methods. 2013; 10(11): 1127–33. PubMed Abstract | Publisher Full Text\n\nShapiro E, Biezuner T, Linnarsson S: Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013; 14(9): 618–30. PubMed Abstract | Publisher Full Text\n\nSierant MC, Choi J: SingleSingle-Cell Ssequencing in Cancer: Recent Applications to Immunogenomics and Multi-omics Tools. Genomics Inform. 2018; 16: e17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDurruthy-Durruthy R, Gottlieb A, Hartman BH, et al.: Reconstruction of the mouse otocyst and early neuroblast lineage at single-cell resolution. Cell. 2014; 157(4): 964–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAchim K, Pettit JB, Saraiva LR, et al.: High-throughput spatial mapping of single-cell RNA-seq data to tissue of origin. Nat Biotechnol. 2015; 33(5): 503–9. PubMed Abstract | Publisher Full Text\n\nSatija R, Farrell JA, Gennert D, et al.: Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015; 33(5): 495–502. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaraiskos N, Wahle P, Alles J, et al.: The Drosophila embryo at single-cell transcriptome resolution. Science. 2017; 358(6360): 194–199. PubMed Abstract | Publisher Full Text\n\nFowlkes CC, Hendriks CL, Keränen SV, et al.: A quantitative spatiotemporal atlas of gene expression in the Drosophila blastoderm. Cell. 2008; 133(2): 364–74. PubMed Abstract | Publisher Full Text\n\nAlonso A, Carrea A, Diambra L: Prediction of cell position using single-cell transcriptomic data: an iterative procedure. 2019. http://www.doi.org/10.5281/zenodo.3470061"
}
|
[
{
"id": "55476",
"date": "24 Oct 2019",
"name": "Roosevelt Alves Da Silva",
"expertise": [
"Reviewer Expertise Biological physics",
"computational science",
"protein 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\nIn their manuscript entitled “Prediction of cell position using single-cell transcriptomic data: an iterative procedure ” Alonso et al. apply a method to predict the cell position combining single-cell transcriptomic data with a reference atlas. The study is submitted as part of the SCTC DREAM Challenge, which aims to evaluate different algorithms for this prediction task using 20, 40 or 60 genes as reference. The authors test their approach on SCTC challenge data-sets. They have three improvements on a method previously published (ref 71): gene selection, a new scoring function, and an iterative procedure for improve the predictions. The paper is generally well-written, with a concise description of the problem and challenge. However it has room for improvement, especially in the manner information is presented. My criticisms are related to the clarification of details I thought to be missing from Methods and Results sections.\n\nMajor comments:\nI suggest to authors to add a sentence that help to reader to understand the aim of the paper in the Introduction.\n\nThe fluxogram of Fig.3 seems has not a initial point. Why? I suggest to authors divide the procedure in layers. Also authors must give a description in the legend of this figure.\n\nPanel A of Fig. 4 has not legend, please add one.\nMinor comments:\nThe description of acronyms MCC is duplicated. The author must introduce it in the first time that it is mentioned.\n\nPlease, add adequate references for Mathews correlation coefficient (in Methods) and for Jaccard distance (in Discussion).\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": "5375",
"date": "09 Apr 2020",
"name": "Luis Diambra",
"role": "Author Response",
"response": "Response: We would like to thank Referee 1 for reviewing our paper, and for his/her positive comments. Below, we give him/her a point by point response for all his/her comments. Major comments: I suggest to authors to add a sentence that help to reader to understand the aim of the paper in the Introduction. Response: We agree. We have added two sentences at the end of the Introduction in order to clarify the aim of our work. The fluxogram of Fig.3 seems has not a initial point. Why? I suggest to authors divide the procedure in layers. Also authors must give a description in the legend of this figure. Response: In this new version of the manuscript we have added a description to Fig. 3, and modified its initial point and the layers in order to make them clearer. Panel A of Fig. 4 has not legend, please add one. Response: In this new version of the manuscript we have added a legend to Fig. 4A. Minor comments: The description of acronyms MCC is duplicated. The author must introduce it in the first time that it is mentioned. Response: The referee is correct, we have already fixed it. Please, add adequate references for Mathews correlation coefficient (in Methods) and for Jaccard distance (in Discussion). Response: We have added two new references: ref10: 10.1016/0005-2795(75)90109-9 and ref11: 10.1038/234034a0, respectively."
}
]
},
{
"id": "55478",
"date": "08 Nov 2019",
"name": "Pablo Meyer",
"expertise": [
"Reviewer Expertise Computational biology and systems 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\nAlonso et al propose a nice idea for solving the gene to position problem posed in the DREAM single cell transcriptomics challenge, but at the end fall short while finding ways to improve their pipeline. The explanations of why they used such approach are also scarce. Although maybe obvious, it would be important that they explain why they chose genes with maximum entropy. why 100 outgroup genes, how the values of w1 and w2 were selected and why doing a 2 step optimization. Overall the rationale of their approach should be summarized in a couple of sentences in order to make it clearer, unfortunately the diagram does not clarify the rationale either. Also, given that this criteria did not improve significantly while iterating, they should have tried other selection criteria such as high expression level, or maybe other metrics such as the ones proposed in the challenge. Maybe trying to optimize the position and the gene expression correlation as interlocked steps is the source of the problem. Some discussion regarding this should be added.\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": "5376",
"date": "09 Apr 2020",
"name": "Luis Diambra",
"role": "Author Response",
"response": "Alonso et al propose a nice idea for solving the gene to position problem posed in the DREAM single cell transcriptomics challenge, but at the end fall short while finding ways to improve their pipeline. Response: We would like to thank the Referee 2 for reviewing our paper, and for his positive comments. Below we gives him a point by point response for all his comments. The explanations of why they used such approach are also scarce. Although maybe obvious, it would be important that they explain why they chose genes with maximum entropy. why 100 outgroup genes, how the values of w1 and w2 were selected and why doing a 2 step optimization. Response: The approach for our gene set selection has two steps. We agree that these steps have not been justified in our previous version. Now we have added a rationale for our procedure. It is well known that many genes co-express, that is, their expression profiles are highly correlated. This correlation introduces a degree of redundancy in the expression matrix, which is oftenly reduced by clustering those genes with similar expression profiles. We assume that the genes within each cluster would not provide much more information than just one in this group. Since the challenge requires choosing a small set of genes, we must choose one gene that represents each cluster. The question is then, which one? This selection is made with the second criterion, which discards those genes with many null values of expression over a large part of the population, because they are associated with distributions with a large peak at zero. Of course, one could also use a criterion that selects the gene with higher expression level, as you proposed (see last response). Regarding the number of genes used as the outgroup genes, we did not make any computation to determine this number, or to answer if there is an optimal number of genes. We just chose a number of genes greater than the 86 genes, which seems to be good enough for the correct prediction of the cell positions. Regarding the values of w1 and w2, unfortunately we cannot imagine other method to determine w1 and w2 different from the brute force. In this sense, we began our study with a value of 0.5 for each weight. That gave quite bad results. Then, we increased the weight w1 and decreased w2 by 0.10 at each step, obteining improvements in the performance, until we reached the weights shown in the paper. We did not try to find the better weitghs. Initially, we assumed that an iterative procedure could be a kind of self-consistent method that would lead to an improvement in performance at every step. Unfortunately, that was not verified. The iterative procedure improved the performance only in the first steps. For this resubmission, we have made several modifications and performed perdictions, testing other hypotheses. However, we did not manage to substantially improve our own results (see the last point). Overall the rationale of their approach should be summarized in a couple of sentences in order to make it clearer, unfortunately the diagram does not clarify the rationale either. Response: We have remade the diagram of Fig. 3, and we have added a short description to each step in the legend of this figure. Also, given that this criteria did not improve significantly while iterating, they should have tried other selection criteria such as high expression level, or maybe other metrics such as the ones proposed in the challenge. Maybe trying to optimize the position and the gene expression correlation as interlocked steps is the source of the problem. Some discussion regarding this should be added. Response: We have evaluated the performance of the approach by considering several modifications. In particular, those proposed by the referee: 1.- We selected the genes with higher expression levels within each cluster, instead of the genes with broadest level distribution. This gene selection strategy gave similar results to the one presented in the original version. 2.- We used the metrics based only on MCC, instead of the combination of two metrics. In this case, the predicted expression patterns computed in the first iteration are used to compute the MCC of the of the outgroup genes with the corresponding expression level of each one of the 1297 single cells. Finally, we computed a composed score S, defined as S = w1* c + w2* o, where c is MCC score computed in the first step (with the 20 genes), and o is the MCC score computed with the outgroup set of genes, and w1 and w2 are the respective weights. We do not understand the reasons why subsequent iterations do not improve performance, significantly. However, we believe that the predicted expression pattern of the outgroup set of genes could be used to improve, in subsequent steps, any other method with better performance than the one we have proposed."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1775
|
https://f1000research.com/articles/8-794/v1
|
06 Jun 19
|
{
"type": "Research Article",
"title": "Post-Ebola Community Health Worker programme performance in Kenema District, Sierra Leone: A long way to go!",
"authors": [
"Harold Thomas",
"Katrina Hann",
"Mohamed Vandi",
"Joseph Bengalie Sesay",
"Koi Sylvester Alpha",
"Robinah Najjemba",
"Katrina Hann",
"Mohamed Vandi",
"Joseph Bengalie Sesay",
"Koi Sylvester Alpha",
"Robinah Najjemba"
],
"abstract": "Background: The devastating 2014-2015 Ebola outbreak in Sierra Leone could erode the gains of the health system including the Community Health Worker (CHW) programme. We conducted a study to ascertain if the positive trend in reporting cases of malaria, pneumonia and diarrhoea treated by CHWs in the post-Ebola period has been sustained 18 months post-Ebola. Methods: We conducted a retrospective cross-sectional study using aggregated CHW programme data (2013-2017) from all Primary Health Units in Kenema district. Data was extracted from the District Health Information System and analysed using STATA. Data in the pre- (June 2013-April 2014), during- (June 2014-April 2015) and post-Ebola recovery (June 2016-April 2017) periods was compared and analysed for reporting completeness; Rapid Diagnostic Tests (RDTs) performed and cases of malaria, diarrhoea and pneumonia treated per month. Differences across periods were tested using two-sample t-test with significance set at 0.05. Results: CHW reporting increased from pre-Ebola by 8% (p-value=0.29) intra-Ebola and 19% (p-value=0.012) post-Ebola. Compared to the pre-Ebola period, in the post-Ebola recovery period, there was a significant increase in the mean monthly reported RDTs of 35% (p-value=0.020); malaria treatments 66% (p-value<0.001); and pneumonia treatments increased by 80% (p-value=0.004). Conversely, the mean monthly diarrhoea cases treated decreased by 20% (p-value=0.16) in the post-Ebola period. Conclusion: The resiliency demonstrated by the CHW programme during and immediately after the Ebola outbreak has been sustained in the post-Ebola recovery period. Continued programme investments in supportive supervision and financial incentives for CHWs will be critical to ensure uninterrupted contribution towards Sustainable Development Goal 3.",
"keywords": [
"Universal Health Coverage",
"Sustainable Development Goals",
"health systems",
"SORT IT",
"operational research"
],
"content": "Introduction\n\nThe effect of the 2014–2015 West African Ebola outbreak on Community Health Worker (CHW) services in Kenema district, Sierra Leone, was assessed through a retrospective cross-sectional study comparing CHW reporting and services before (June 2013-April 2014), during (June 2014-April 2015) and post-outbreak (November 2015-April 2016)1. The study found CHW reporting completeness and reported treatments for malaria increased post-Ebola, while those for pneumonia and diarrhoea returned to pre-outbreak levels. Results showed CHWs stopped performing Rapid Diagnostic Tests (RDTs) during the Ebola period and did not resume until after the outbreak. The study recommended additional investigations into preliminary positive trends in the early post-Ebola period to determine if these post-outbreak tendencies represent a resumption of pre-Ebola CHW programme performance or a continued progression. Therefore, we aimed to understand whether health system performance was sustained 18 months after the outbreak by reporting the trend and comparing CHW system performance (reporting completeness, reported diagnosis and treatment services among reports received) in Kenema district in the pre-, intra- and post-Ebola recovery periods for children under five years.\n\n\nMethods\n\nThis was a retrospective cross-sectional study using aggregate CHW programme data from Kenema district, Sierra Leone. Details of Kenema and the CHW programme were previously described1. The study population included all CHW programme reports from Kenema Peripheral Health Units (PHUs) during the pre-(June 2013-April 2014), intra-(June 2014-April 2015), and post-Ebola recovery (June 2016-April 2017) periods. Data during the month of May 2014 were excluded to prevent potential spill-over effects across periods. Consistent months were deliberately chosen to allow for seasonal comparison across the three periods.\n\nCHW programme data were extracted from the electronic Ministry of Health and Sanitation (MoHS) District Health Information Systems (DHIS2), which is aggregated from monthly summary sheets submitted by PHU supervisors. Data on the following variables were extracted per month: facility reporting completeness; reported malaria RDTs; treatment for malaria, diarrhoea and pneumonia. In this study we defined CHW reporting completeness as number of facilities with fully (100%) complete reporting for CHW monthly summary sheets over the total number of expected facility reports. We imported data into STATA v14.22. We produced descriptive summary statistics and examined statistical differences between periods using two-sample t-test, with significance set at 0.05.\n\nThe Sierra Leone Ethics and Scientific Review Committee (dated 18 December 2018) and the Ethics Advisory Group of the International Union Against Tuberculosis Lung Disease (UAG number 65/18) provided ethics and institutional approval. Since aggregate programme data were used, participant consent was not sought.\n\n\nResults\n\nFigure 1 shows the proportion of facilities with CHW complete reports monthly in the pre-, intra- and post-Ebola recovery periods. The number of PHUs submitting complete CHW reports ranged between 39–129 (27–89%) and 98–134 (68–92%) in the pre- and intra-outbreak periods respectively. Conversely, in the recovery period, 136 (94%) facilities consistently completed reports each month.\n\nCHW – community health worker. Pre-Ebola Period – June 2013 – April 2014, Intra-Ebola Period – June 2014 – April 2015, Post-Ebola Recovery Period – June 2016 – April 2017.\n\nFigure 2 shows trends across pre-, intra- and post-Ebola recovery periods for CHW programme performance indicators. Table 1 shows that the mean monthly reported RDTs had an increasing trend in the pre-, intra- and post-Ebola recovery periods, with increases of 11% intra-Ebola and 35% (p-value=0.012) post-Ebola compared to the pre-Ebola period. The mean monthly malaria treatments reported trends similar to RDTs with increases of 31% (p-value=0.026) and 66% (p-value=0.020) intra-Ebola and post-Ebola, respectively.\n\nRDT - Rapid Diagnostic Test. CHW – community health worker. Pre-Ebola Period – June 2013 – April 2014. Intra-Ebola Period – June 2014 – April 2015. Post-Ebola Recovery Period – June 2016 – April 2017.\n\nCI: confidence interval; RDT: rapid diagnostic test.\n\nPre-Ebola: June 2013 – April 2014; Intra-Ebola: June 2014 – April 2015; Post-Ebola Recovery: June 2016 – April 2017.\n\nThe mean monthly diarrhoea treatments reported exhibited a downward trend, in the pre- and intra-Ebola periods, followed by a small uptick in the post-Ebola recovery period. However, compared to the pre-Ebola period, overall monthly mean reported treatments significantly decreased by 42% (p-value=0.013) in the intra-Ebola, while the reduction in the post-Ebola recovery period was not statistically significant (p-value=0.16).\n\nThe mean reported monthly pneumonia treatments declined by 11% intra-Ebola but grossly increased by 80% (p-value=0.004) during the recovery period compared to the pre-Ebola period (Table 1).\n\n\nDiscussion\n\nOur study results indicate a general trend of the CHW programme in Kenema district sustaining comparable performance levels into the post-Ebola recovery period as compared to pre-Ebola for reporting completeness and reported RDTs, malaria and pneumonia treatments. Comparable to results from the previous study1 and the region3, this may be due to sustained investment in key areas affecting supply and services. The Integrated Disease Surveillance and Response approach, which emphasises active community-based surveillance and completeness of reporting, was strengthened in the intra-Ebola period3. In addition, supportive supervision, a critical element to the success of CHW programmes4, was revived after the outbreak3. Finally, financial incentives for CHWs initiated during the outbreak were subsequently incorporated into the national policy in 20165. Furthermore, the CHWs have reported increased community awareness of signs and symptoms of common childhood illnesses, translating into increased demand for services3.\n\nWe found fewer missing reports in the pre- and intra-Ebola periods compared to the previous study2, most likely due to retrospective data entry into the DHIS2. While we found higher mean reported RDTs performed in the post-Ebola period, we still observed fewer in the intra-Ebola period corresponding with the enactment of the “no touch policy,” similar to the previous report1,3,6.\n\nOur results reveal an absolute decline in the reported diarrhoea treatments during the recovery period. The promotion of hygiene practices through community sensitization7 and institution of bye-laws by community stakeholders in the intra-Ebola period may have had lasting effects on behaviour and thus contributing to the reduction in reported diarrhoea treatments during the post-Ebola period3. However, the difference in reported treatments between pre- and post-Ebola periods was not found to be significant, so this trend may warrant future investigation.\n\nUtilising pre-Ebola service levels for comparisons, while useful, still reflect benchmarks of weak systems functioning, a factor which contributed to the impact of the Ebola outbreak itself8. Therefore, it is imperative that such comparisons be evaluated in this light. Therefore, in order to achieve the vision of the Sustainable Development Goal (SDG) for good health and wellbeing beyond pre-Ebola-level benchmarks, sustained investments in supportive supervision and financial incentives for CHWs are essential for the programme.\n\nA strength of the study is the use of complete district PHU data for the study period. Furthermore, we adhered to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines for the reporting of observational data and sound ethical principles9. Primary weaknesses of our study were the use of routinely collected data, which was influenced by reporting completeness, thus restricting reporting of morbidity; lack of data triangulation; inability to validate the electronic data base against the raw data and to generalise to the national CHW programme.\n\nIn conclusion, although our study established a sustained trend towards the pre-Ebola CHW service levels 18 months after the outbreak, there is need for continued investment in the CHW programme to continue gains in programme performance in order to contribute towards SDG 3.\n\n\nData availability\n\nOpen Science Framework: Thomas_Harold_SORTIT2_CHW_data 2019. https://doi.org/10.17605/OSF.IO/2S83W10.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nThe Sierra Leone Health Management Information Systems, the District Health Information System 2 (DHIS2), is accessible with a Ministry of Health and Sanitation (MOHS) login through https://sl.dhis2.org/. The Directorate of Policy, Planning, and Information (DPPI) can be contacted to arrange access through Dr. Francis Smart (drfsmart@gmail.com), Director, DPPI, MOHS.",
"appendix": "Grant information\n\nThe programme was funded by the Special Programme for Research and Training in Tropical Diseases hosted at the World Health Organization (TDR).\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\nThis research was conducted through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization (WHO/TDR) and implemented with partners. The training model is based on a course developed jointly by the International Union Against Tuberculosis and Lung Disease (The Union) and Medécins sans Frontières (MSF). The specific SORT IT programme which resulted in this publication was jointly developed and implemented by: WHO/TDR, the Sierra Leone Ministry of Health and Sanitation, WHO Sierra Leone, the Centre for Operational Research, The Union, Paris, France; the Alliance for Public Health, Ukraine; the Institute of Tropical Medicine, Antwerp, Belgium; and Sustainable Health Systems, Freetown, Sierra Leone.\n\n\nReferences\n\nVandi MA, van Griensven J, Chan AK, et al.: Ebola and community health worker services in Kenema District, Sierra Leone: please mind the gap! Public Health Action. 2017; 7(Suppl 1): S55–S61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStataCorp: Stata Statistical Software: Release 14. College Station, TX: StataCorp LP; 2015.\n\nMiller NP, Milsom P, Johnson G, et al.: Community health workers during the Ebola outbreak in Guinea, Liberia, and Sierra Leone. J Glob Health. 2018; 8(2): 020601. PubMed Abstract | Free Full Text\n\nNdima SD, Sidat M, Give C, et al.: Supervision of Community Health Workers in Mozambique: a qualitative study of factors influencing motivation and programme implementation. Hum Resour Health. 2015; 13(1): 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Health and Sanitation (MoHS): National Community Health Worker Policy 2016-2020. Freetown: MoHS; 2016; 34. Reference Source\n\nUNICEF and World Health Organization: Guidelines for a revised implementation of Integrated Community Case Management of Childhood Illnesses (ICCM) during the Ebola outbreak. UNICEF, WHO; 2014; 3. Reference Source\n\nFOCUS 1000, US Centers for Disease Control and Prevention, UNICEF: Follow-up study on public knowledge, attitudes, and practices relating to Ebola virus disease prevention and medical care in Sierra Leone (Ebola KAP-4), Preliminary Report. Freetown, Sierra Leone: FOCUS 1000; 2015; 21.\n\nO'Hare B: Weak health systems and Ebola. Lancet Glob Health. 2015; 3(2): e71–2. PubMed Abstract | Publisher 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\nThomas H: Thomas_Harold_SORTIT2_CHW_data. osf.io/2s83w. 2019. http://www.doi.org/10.17605/OSF.IO/2S83W"
}
|
[
{
"id": "49603",
"date": "24 Jun 2019",
"name": "Palanivel Chinnakali",
"expertise": [
"Reviewer Expertise Operational Research (Implementation Research)",
"Tuberculosis",
"HIV/AIDS"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is written well.\nIt will be better to provide some more background about the scale of involvement of CHWs during Ebola outbreak. How CHWs were involved, time points when the Ebola cases came down, post outbreak responsiblities for CHWs related to Ebola\n\nImpact/severity of Ebola outbreak in the study district Kenema compared to others.\n\nStudy design: Instead of retrospective cross sectional study, I suggest this 'We performed trend analysis using routinely reported aggregate CHW programme data'\n\nMethods: Provide information on the total population of the district, malaria and diarrhea statistics, number of health facilities, number of CHWs present during the pre-Ebola period, how they report (paper based/electronic).\n\nMissing reports: present the facts in the results section and then can be brought under 'discussion' section\n\nReporting on number of CHWs would be good in the results section. Were there any deaths among CHWs due to Ebola?\n\nWhat could be the possible reasons for 80% increase in reporting of Pneumonia during post Ebola period? It would be good to discuss the possible reasons\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5371",
"date": "09 Apr 2020",
"name": "Katrina Hann",
"role": "Author Response",
"response": "RE: Post-Ebola Community Health Worker programme performance in Kenema District, Sierra Leone: A long way to go! [version 1; peer review: 1 approved, 1 approved with reservations] We have carefully read through the peer review and have revised our manuscript accordingly. We see the manuscript as improved as a result of this peer review process, and thank you for your support in taking the process to this stage.Please find for your consideration the following: A point-by-point response to the comments and suggestions of the reviewers in red font. A new revised version of the manuscript, in tracked changes A new, revised version of the manuscript, no tracked changes. We hope that these modifications meet with your favourable review. Please do not hesitate to request any further changes. Kind regards, Harold ThomasDirectorate of Health Security and EmergenciesMinistry of Health and Sanitation (MoHS)FreetownSierra Leone Author Response to Reviewer Report for Version 1 from Palanivel Chinnakali 24 June 2019 Thank you for your thoughtful review of the manuscript and your insightful suggestions. We have responded as below. 1. Reviewer comment:The manuscript is written well. It will be better to provide some more background about the scale of involvement of CHWs during Ebola outbreak. How CHWs were involved, time points when the Ebola cases came down, post outbreak responsiblities for CHWs related to Ebola.Response:Thank you very much for your comments. We agree that additional comment on the CHW programme will be useful to the reader. We have added further details the methods study setting section.2. Reviewer comment:Impact/severity of Ebola outbreak in the study district Kenema compared to others. Response:Thank you for your comment. Your suggestion is interesting, However, this is beyond the scope of our study as we did not employ comparative methods.3. Reviewer comment:Study design: Instead of retrospective cross sectional study, I suggest this 'We performed trend analysis using routinely reported aggregate CHW programme data'.Response:Thank you for your suggestion. We agree with your point and have added in the methods section a description of the trend analysis over the stipulated periods.4. Reviewer comment:Methods: Provide information on the total population of the district, malaria and diarrhea statistics, number of health facilities, number of CHWs present during the pre-Ebola period, how they report (paper based/electronic).Response:Thank you for your comment. We do not have data on the number of CHWs operating during the period under study. However, we did estimate the number of CHWs based off of population data for the district from the 2015 Census and an estimate of the total number of CHWs nationwide from the Human Resources for Health Strategy 2017. We have clarified this in the manuscript, with associated references. 5. Reviewer comment:Missing reports: present the facts in the results section and then can be brought under 'discussion' section. Response:Thank you very much for your comment. We appreciate this and we have clarified this in the results section. We already mentioned reporting completeness as a weakness of the study in the discussion section, and, therefore, have not made adjustments.6. Reviewer comment:Reporting on number of CHWs would be good in the results section. Were there any deaths among CHWs due to Ebola?Response:Thank you for your comments. Whilst we appreciate your comments, we have addressed the issue of data availability with regards to number of CHWs, and the mortality of CHWs is outside the scope of our study.7. Reviewer comment:What could be the possible reasons for 80% increase in reporting of Pneumonia during post Ebola period? It would be good to discuss the possible reasons. Response:Thank you for your comments. We have addressed that in the discussion section in the paragraph where we introduce possible reasons for increase in service demand."
}
]
},
{
"id": "51795",
"date": "13 Aug 2019",
"name": "Joanna Raven",
"expertise": [
"Reviewer Expertise Social science",
"health systems."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study which investigates the CHW system performance pre, during and post Ebola periods looking at CHW reporting, reported diagnosis and treatment services. It particularly looks at sustained trends of performance 18 months after the end of Ebola. The article is well written and presented.\n\nI suggest the following improvements to the paper:\nIn the introduction, a more detailed description of the CHW programme pre-Ebola, during Ebola and post Ebola is needed to provide context to the study and the findings. For example, who provided the supplies for the RDT and treatments, the financial incentives for the CHWs, and the supervision? How was this funded? Then in the discussion, this can be drawn upon to discuss the findings and offer recommendations.\n\nFinancial incentives for CHWs are included in the national CHW policy 2016-2020. However, there have been issues with getting the incentives to the CHWs, with many CHWs not receiving them. So, a discussion of the role of incentives in CHW performance should be discussed - is it the promise of money that motivates CHWs? How long will this willingness last, if the payments do not materialise?\n\nThe definition of the reporting completeness is unclear. and needs revision. It is unclear whether completeness indicates the reports from ALL the CHWs within the catchment area of the facility. If not, then how this affects the figures for reporting cases and treatment, needs to be discussed.\n\nReduction in diarrhoea treatment trend needs to be explored more, for example, comparisons made with diarrhoea treatment rates in other areas of Sierra Leone or national rates, and changes in availability of drugs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5372",
"date": "09 Apr 2020",
"name": "Katrina Hann",
"role": "Author Response",
"response": "We have carefully read through the peer review and have revised our manuscript accordingly. We see the manuscript as improved as a result of this peer review process, and thank you for your support in taking the process to this stage.Please find for your consideration the following: A point-by-point response to the comments and suggestions of the reviewers in red font. A new revised version of the manuscript, in tracked changes A new, revised version of the manuscript, no tracked changes. We hope that these modifications meet with your favourable review. Please do not hesitate to request any further changes. Kind regards, Harold ThomasDirectorate of Health Security and EmergenciesMinistry of Health and Sanitation (MoHS)FreetownSierra Leone Point-by-point response to the reviewer suggestionsAuthor Response to Reviewer Report for Version 1 from Joanna Raven 13 Aug 2019 Thank you for your thoughtful review of the manuscript and your suggestions. We have responded as below. 1. Reviewer comment:This is an interesting study which investigates the CHW system performance pre, during and post Ebola periods looking at CHW reporting, reported diagnosis and treatment services. It particularly looks at sustained trends of performance 18 months after the end of Ebola. The article is well written and presented. I suggest the following improvements to the paper:In the introduction, a more detailed description of the CHW programme pre-Ebola, during Ebola and post Ebola is needed to provide context to the study and the findings. For example, who provided the supplies for the RDT and treatments, the financial incentives for the CHWs, and the supervision? How was this funded? Then in the discussion, this can be drawn upon to discuss the findings and offer recommendations.Response:We have added in a more detailed description of the CHW programme and its operations in the study setting section under methods.2. Reviewer comment:Financial incentives for CHWs are included in the national CHW policy 2016-2020. However, there have been issues with getting the incentives to the CHWs, with many CHWs not receiving them. So, a discussion of the role of incentives in CHW performance should be discussed - is it the promise of money that motivates CHWs? How long will this willingness last, if the payments do not materialise?Response:We agree that the implementation of distribution of financial incentives for CHWs may be challenging. However, we see a discussion on the role of incentives in CHW performance as beyond the scope of this paper.3. Reviewer comment:The definition of the reporting completeness is unclear. and needs revision. It is unclear whether completeness indicates the reports from ALL the CHWs within the catchment area of the facility. If not, then how this affects the figures for reporting cases and treatment, needs to be discussed.Response:We have revised our description of the definition of reporting completeness in the manuscript. In addition, we have added this point to our discussion section.4. Reviewer comment:Reduction in diarrhoea treatment trend needs to be explored more, for example, comparisons made with diarrhoea treatment rates in other areas of Sierra Leone or national rates, and changes in availability of drugs.Response:We agree with these points, but do not have data in the context of this study by which to make such comparisons. We have, however, pointed to the need for future investigations that may further explain this finding."
}
]
}
] | 1
|
https://f1000research.com/articles/8-794
|
https://f1000research.com/articles/9-129/v1
|
21 Feb 20
|
{
"type": "Brief Report",
"title": "Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CLpro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates",
"authors": [
"Yu Wai Chen",
"Chin-Pang Bennu Yiu",
"Kwok-Yin Wong",
"Chin-Pang Bennu Yiu"
],
"abstract": "We prepared the three-dimensional model of the SARS-CoV-2 (aka 2019-nCoV) 3C-like protease (3CLpro) using the crystal structure of the highly similar (96% identity) ortholog from the SARS-CoV. All residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. Comparison of the polyprotein PP1AB sequences showed 86% identity. The 3C-like cleavage sites on the coronaviral polyproteins are highly conserved. Based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the SARS-CoV enzyme can be conferred on its SARS-CoV-2 counterpart. With the 3CLpro molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. Among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache. The drugs Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes.",
"keywords": [
"COVID-19",
"SARS",
"2019-nCoV",
"3C-like protease",
"drug repurpose",
"antiviral",
"coronavirus",
"virtual screening",
"molecular modelling",
"ledipasvir",
"velpatasvir",
"Hepatitis C virus",
"HCV"
],
"content": "Introduction\n\nOn 7 January 2020, a new coronavirus, 2019-nCoV (now officially named SARS-CoV-2) was implicated in an alarming outbreak of a pneumonia-like illness COVID-19, originating from Wuhan City, Hubei, China. Human-to-human transmission was first confirmed in Guangdong, China1. The World Health Organisation has declared this a global public health emergency — on 15 February 2020, there are more than 65,000 confirmed cases reported, and the death toll is over 1500. In the height of the crisis, this virus is spreading at a rate and scale far worse than previous coronaviral epidemics.\n\nIt was immediately evident from its genome that the coronavirus is evolutionarily related (80% identity) to the beta-coronavirus implicated in the severe acute respiratory syndrome (SARS), which originated in bats and was causative of a global outbreak in 2003. The momentum of research on developing antiviral agents against the SARS-CoV carried on after the epidemic subsided. Despite this, no SARS treatment has yet come to fruition; however, knowledge acquired from the extensive research and development efforts may be of use to inform the current therapeutic options.\n\nThe viral genome encodes more than 20 proteins, among which are two proteases (PLpro and 3CLpro) that are vital to virus replication; they cleave the two translated polyproteins (PP1A and PP1AB) into individual functional components. The 3-chymotrypsin-like protease (3CLpro, aka main protease, Mpro) is considered to be a promising drug target. Tremendous effort has been spent on studying this protein in order to identify therapeutics against the SARS-CoV in particular and other pathogenic coronaviruses (e.g. MERS-CoV, the Middle East respiratory syndrome coronavirus) in general because they share similar active sites and enzymatic mechanisms. The purpose of this study is to build a molecular model of the 3CLpro of the SARS-CoV-2 and to carry out virtual screening to identify readily usable therapeutics. It was not our intention, however, to comment on other structure-based drug design research as these will not be timely for the current epidemic.\n\n\nMethods\n\nThe translated polyprotein (PP1AB) sequence was obtained from the annotation of the GenBank entry of the SARS-CoV-2 genome (accession number MN908947). By comparing this sequence with the SARS-CoV PP1AB sequence (accession number ABI96956), the protease cleavage sites and all mature protein sequences were obtained. Sequence comparison and alignment were performed with BLASTp.\n\nThe high-resolution apo-enzyme structure of SARS-CoV 3CLpro (PDBID: 2DUC)2 was employed as the template. The variant residues were “mutated” in silico by SCWRL43, followed by manual adjustment to ensure that the best side-chain rotamer was employed (Table 2). The rebuilt model was subjected to steepest descent energy minimisation by Gromacs 2018.4 using the Gromos 54A7 forcefield, with a restraint force constant of 1000 kJ mol-1 nm-2 applied on all backbone atoms and all atoms of the vital residues (Table 1). Accessible surface area of residues were calculated with areaimol of the CCP4 suite v7.0.\n\nThe 12 variant residues with reference to the SARS-CoV enzyme are shown with the respective treatment of rotamer. “A” and “B” refers to the individual chains of the dimeric model. Both chains are in the crystal asymmetric unit and are not identical. The rotamer symbol (bracketed) is defined according to the conventions of Richardson15, followed by its respective rank of popularity. ASA: accessible surface area (average of A and B chains) of the residue in the SARS-CoV 3CLpro structure, in Å2 and in % relative to the ASA of a residue X in the Gly-X-Gly conformation.\n\nMTiOpenScreen web service4 was used for screening against its library of 7173 purchasable drugs (Drugs-lib), with the binding site grid specified by the active-site residues. The active sites on chain A and chain B were screened independently with AutoDock Vina5. When the crystal structure was released, it was stripped of its inhibitor and subjected to a screening. A list of 4,500 target:ligand docking combinations ranked by binding energies was produced for each screen. The top 10 or 11 (ranked using a binding energy cut-off) hits for chains A and B were examined visually in PyMOL (version 1.7.X)6.\n\nAn earlier version of this article can be found on ChemRxiv (DOI: 10.26434/chemrxiv.11831103.v2).\n\n\nResults\n\nThe first available genome was GenBank MN908947, now NCBI Reference Sequence NC_045512. From it, the PP1AB sequence of SARS-CoV-2 was extracted and aligned with that of SARS-CoV. The overall amino-acid sequence identity is very high (86%). The conservation is noticeable at the polyprotein cleavage sites. All 11 3CLpro sites2 are highly conserved or identical (Extended data7, Table S1), inferring that their respective proteases have very similar specificities. The 3CLpro sequence of SARS-CoV-2 has only 12 out of 306 residues different from that of SARS-CoV (identity = 96%).\n\nWe compared the polyprotein PP1AB and the 3CLpro sequences among all 11 SARS-CoV-2 genomes (GenBank MN908947, MN938384, MN975262, MN985325, MN988668, MN988669, MN988713, MN994467, MN994468, MN996527 and MN996528) that were available on 1 February 2020. With reference to MN908947 (NC_045512), among the 7096 residues, there is only one variable residue in each of MN975262 (in NSP-4), MN994467 (in NSP-2), MN994468 (in NSP-13), MN996527 (in NSP-16); and two in MN988713 (in NSP-1 and NSP-3). The remaining five have no difference. To summarise, all SARS-CoV-2 3CLpro sequences and all their cleavage junctions on their polyproteins are 100% conserved.\n\nThe amino acids that are known to be important for the enzyme’s functions are listed in Table 1. Not unexpectedly, none of the 12 variant positions are involved in major roles. Therefore, we are confident to prepare a structural model of the SARS-CoV-2 3CLpro by molecular modelling (Extended data7, Figure S1), which will be immediately useful for in silico development of targeted treatment. After we submitted the first draft of this study, the crystal structure of SARS-CoV-2 3CLpro was solved and released (PDB ID 6LU7), which confirms that the predicted model is good within experimental errors (Extended data7, Figure S2).\n\nWhen examined in molecular graphics6, all solutions were found to fit into their respective active sites convincingly. The binding energies of chain A complexes were generally higher than those of chain B by approximately 1.4 kcal mol-1 (Table 3). This presumably demonstrates the intrinsic conformational variability between the A- and B-chain active sites in the crystal structure (the average root-mean-square deviation (rmsd) in Cα atomic positions of active-site residues is 0.83 Å). In each screen, the differences in binding energies are small, suggesting that the ranking is not discriminatory, and all top scorers should be examined. We combined the two screens and found 16 candidates which give promising binding models (etoposide and its phosphate counted as one) (Table 3).\n\nThe left and right columns are the results of A and B chains, respectively. The top scorers are listed first, then the equivalent top scorers of the other chain listed at the lower half. B.E.: AutoDock Vina binding energy in kcal mol-1. The number of hits of a drug is the times it appears among all results within a screen regardless of rank, only the binding energy of the top-ranking hit was shown. Etoposide and its phosphate are listed separately in the screens. Approved and pre-approved drugs are shown in green and orange, respectively. Except dihydroergocristine and ditercalinium, all approved drugs have undergone post-market surveillance, i.e. Phase 4.\n\nWe checked the actions, targets and side effects of the 16 candidates. Among these, we first noticed velpatasvir (Figure 1A, D) and ledipasvir, which are inhibitors of the NS5A protein of the hepatitis C virus (HCV). Both are marketed as approved drugs in combination with sofosbuvir, which is a prodrug nucleotide analogue inhibitor of RNA-dependent RNA polymerase (RdRp, or NS5B). Interestingly, sofosbuvir has recently been proposed as an antiviral for the SARS-CoV-2 based on the similarity between the replication mechanisms of the HCV and the coronaviruses14. Our results further strengthen that these dual-component HCV drugs, Epclusa (velpatasvir/sofosbuvir) and Harvoni (ledipasvir/sofosbuvir), may be attractive candidates to repurpose because they may inhibit two coronaviral enzymes. A drug that can target two viral proteins substantially reduces the ability of the virus to develop resistance. These direct-acting antiviral drugs are also associated with very minimal side effects and are conveniently orally administered (Table 4).\n\nDocking of representative drugs into the active sites of A chain (A, B, C) and that of B chain (D, E, F). The catalytic residue surfaces are coloured in yellow. Atom colours of drug: C: cyan; O: red; N: blue; H: white; S: yellow; only polar hydrogens are shown. Prepared with PyMOL.\n\nSources of information: a DrugBank.ca (main), b Wikipedia.org, c ClinicalTrials.gov and d WebMD.com.\n\nThe flavonoid glycosides diosmin (Figure 1B) and hesperidin (Figure 1E), obtained from citrus fruits, fit very well into and block the substrate binding site. Yet, these compounds cause mild adverse reactions (Table 4). Hesperidin hits showed up multiple times, suggesting it has many modes of binding (Figure 1A). Teniposide and etoposide (and its phosphate) are chemically related and turned up in multiple hits with good binding models (Figure 1F). However, these chemotherapy drugs have a lot of strong side effects and need intravenous administration (Table 4). The approved drug venetoclax (Figure 1C) and investigational drugs MK-3207 and R428 scored well in both screens. Venetoclax is another chemotherapy drug that is burdened by side effects including upper respiratory tract infection (Table 4). Not much has been disclosed about MK-3207 and R428.\n\nWe subjected the crystal structure to the same virtual screening procedures. A very similar list of candidates showed up consistently (Extended data7, Table S2) with high scores although ledipasvir was not found.\n\nWe noticed that most of the compounds on the list have molecular weights (MW) over 500, except lumacaftor (MW=452). The largest one is ledipasvir (MW=889). This is because the size of the peptide substrate and the deeply buried protease active site demand a large molecule that has many rotatable dynamics to fit into it.\n\n\nDiscussion\n\nWe identified five trials on ClinicalTrials.gov involving antiviral and immunomodulatory drug treatments for SARS (Table 5), all without reported results; i.e., at present, there are no safe and effective drug candidates against SARS-CoV. This is because once the epidemic is over, there are no patients to recruit for clinical trials. Only the study with streptokinase succeeded in completion of phase 3. It is disappointing that little progress in SARS drug development has been made in the past 17 years. After the 2003 outbreak, numerous inhibitors for the 3CLpro enzyme have been proposed16,17, yet no new drug candidates have succeeded to enter the clinical phase 1.\n\na This covers unknown respiratory viruses. b This includes the COVID-19. Est. = estimated.\n\nOne record which receives a lot of attention amid the current outbreak is the lopinavir/ritonavir combination18. They are protease inhibitors originally developed against HIV. During the 2003 SARS outbreak, despite lacking a clinical trial, they were tried as an emergency measure and found to offer improved clinical outcome18. However, some scientists did express scepticism19. By analogy, these compounds were speculated to act on SARS-CoV 3CLpro specifically, but there is as yet no crystal structure to support that, although docking studies were carried out to propose various binding modes20–23. The IC50 value of lopinavir is 50 μM (Ki = 14 μM) and that for ritonavir cannot be established24. Although this is far from a cure, based on our results that the two CoV 3CLpro enzymes are identical as far as protein sequences and substrate specificities are concerned, we are of the opinion that this is still one of the recommended routes for immediate treatment at the time of writing (early February 2020).\n\nIf we look beyond the 3CLpro, an earlier screen produced 27 candidates that could be repurposed against both SARS-CoV and MERS-CoV25. In addition, the other coronaviral proteins could be targeted for screening. Treatment of the COVID-19 with remdesivir (a repurposed drug in development targeting the RdRp) showing improved clinical outcome has just been reported and clinical trial is now underway26.\n\nWe consider this work part of the global efforts responding in a timely fashion to fight this deadly communicable disease. We are aware that there are similar modelling, screening and repurposing exercises targeting 3CLpro reported or announced20,27–33. Our methods did not overlap, and we share no common results with these studies.\n\n\nData availability\n\nThe 11 SARS-CoV-2 polyprotein PP1AB and 3CLpro sequences used in this study were obtained from NCBI GenBank, accession numbers MN908947 , MN938384, MN975262, MN985325, MN988668, MN988669, MN988713, MN994467, MN994468, MN996527 and MN996528, available on 1 February 2020.\n\nThe SARS-CoV PP1AB sequence wsa obtained from NCBI Protein, accession number ABI96956.\n\nThe two coronavirus protease structures used were obtained from Protein Data Bank, ID 2DUC and 6LU7.\n\nOpen Science Framework: SARS-CoV-2 (2019-nCoV) 3CLpro Model and Screening. https://doi.org/10.17605/OSF.IO/HCU8X7.\n\nThe “Virtual Screening” folder contains the following extended data:\n\n2019-nCoV-3CLpro.pdb. (3D model of the 3CLpro: A and B chains.)\n\nA-screen4500.pdbqt, B-screen4500.pdbqt, X-screen4500.pdbqt. (Virtual screening 3D results of Model A chain, Model B chain and the crystal-structure (A chain) in PDBQT format (can be viewed by any text editor). Use the software PyMOL to open these files. Each result file contains 4500 drug-to-protein docking hits ranked by AutoDock Vina binding energies in kcal mol-1.)\n\nA-screen1500.table.csv, B-screen1500.table.csv, X-screen1500.table.csv. (Virtual screening results (names only) of Model A chain, Model B chain and the crystal-structure (A chain) in CSV format (can be opened by Excel or any text editor). This is a summary of the top 1500 drug-to-protein docking hits ranked by AutoDock Vina binding energies in kcal mol-1.)\n\nThe “Extended Results” folder contains the following extended data:\n\nTab S1.docx (Sequence homology of the 3CLpro cleavage junctions of PP1AB between SARS-CoV-2 and SARS-CoV).\n\nTab S2.docx (The results of virtual screening of drugs on the active site of SARS-CoV-2 3CLpro crystal structure).\n\nFig S1.pptx (The structural model of the SARS-CoV-2 3CLpro protease).\n\nCompare Crystal.docx (A comparison, with Figure S2, of the active sites of model chains A, B and the crystal structure).\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\nChan JF, Yuan S, Kok KH, et al.: A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020; 395(10223): 514–523. PubMed Abstract | Publisher Full Text\n\nMuramatsu T, Takemoto C, Kim YT, et al.: SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity. Proc Natl Acad Sci U S A. 2016; 113(46): 12997–13002. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrivov GG, Shapovalov MV, Dunbrack RL Jr: Improved prediction of protein side-chain conformations with SCWRL4. Proteins. 2009; 77(4): 778–795. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLabbé CM, Rey J, Lagorce D, et al.: MTiOpenScreen: a web server for structure-based virtual screening. Nucleic Acids Res. 2015; 43(W1): W448–W454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010; 31(2): 455–461. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe PyMOL molecular graphics system (Schrödinger, LLC). Reference Source\n\nChen YW: SARS-CoV-2 (2019-nCoV) 3CLpro Model & Screening. 2020. http://www.doi.org/10.17605/OSF.IO/HCU8X\n\nHuang C, Wei P, Fan K, et al.: 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism. Biochemistry. 2004; 43(15): 4568–4574. PubMed Abstract | Publisher Full Text\n\nHsu MF, Kuo CJ, Chang KT, et al.: Mechanism of the maturation process of SARS-CoV 3CL protease. J Biol Chem. 2005; 280(35): 31257–31266. PubMed Abstract | Publisher Full Text\n\nBarrila J, Bacha U, Freire E: Long-range cooperative interactions modulate dimerization in SARS 3CLpro. Biochemistry. 2006; 45(50): 14908–14916. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarrila J, Gabelli SB, Bacha U, et al.: Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CLpro. Biochemistry. 2010; 49(20): 4308–4317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu T, Zhang Y, Li L, et al.: Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure. Virology. 2009; 388: 324–334. PubMed Abstract | Publisher Full Text\n\nChen S, Zhang J, Hu T, et al.: Residues on the dimer interface of SARS coronavirus 3C-like protease: dimer stability characterization and enzyme catalytic activity analysis. J Biochem. 2008; 143(4): 525–536. PubMed Abstract | Publisher Full Text\n\nJu J, Kumara S, Li X, et al.: Nucleotide analogues as inhibitors of viral polymerases. bioRxiv. 2020. Publisher Full Text\n\nLovell SC, Word JM, Richardson JS, et al.: The penultimate rotamer library. Proteins. 2000; 40(3): 389–408. PubMed Abstract | Publisher Full Text\n\nPillaiyar T, Manickam M, Namasivayam V, et al.: An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: peptidomimetics and small molecule chemotherapy. J Med Chem. 2016; 59(14): 6595–6628. PubMed Abstract | Publisher Full Text\n\nKuo CJ, Liang PH: Characterization and inhibition of the main protease of severe acute respiratory syndrome coronavirus. ChemBioEng Reviews. 2015; 2: 118–132. Publisher Full Text\n\nChu CM, Cheng VC, Hung IF, et al.: Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings. Thorax. 2004; 59(3): 252–256. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStockman LJ, Bellamy R, Garner P: SARS: systematic review of treatment effects. PLoS Med. 2006; 3(9): e343. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGruber C, Steinkellner G: Coronavirus COVID-19 (formerly known as Wuhan coronavirus and 2019-nCoV) - what we can find out on a structural bioinformatics level. 2020. Reference Source\n\nDayer MR, Taleb-Gassabi S, Dayer MS: Lopinavir; a potent drug against coronavirus infection: insight from molecular docking study. Arch Clin Infect Dis. 2017; 12(4): e13823. Publisher Full Text\n\nNukoolkarn V, Lee VS, Malaisree M, et al.: Molecular dynamic simulations analysis of ritonavir and lopinavir as SARS-CoV 3CLpro inhibitors. J Theor Biol. 2008; 254(4): 861–867. PubMed Abstract | Publisher Full Text\n\nZhang XW, Yap YL: Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs. Bioorg Med Chem. 2004; 12(10): 2517–2521. PubMed Abstract | Publisher Full Text\n\nWu CY, Jan JT, Ma SH, et al.: Small molecules targeting severe acute respiratory syndrome human coronavirus. Proc Natl Acad Sci U S A. 2004; 101(27): 10012–10017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDyall J, Coleman CM, Hart BJ, et al.: Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob Agents Chemother. 2014; 58(8): 4885–4893. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolshue ML, DeBolt C, Lindquist S, et al.: First case of 2019 novel coronavirus in the United States. N Engl J Med. 2020. PubMed Abstract | Publisher Full Text\n\nXu Z, Peng C, Shi Y, et al.: Nelfinavir was predicted to be a potential inhibitor of 2019-nCov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation. bioRxiv. 2020. Publisher Full Text\n\nLiu X, Wang Xj: Potential inhibitors for 2019-nCoV coronavirus M protease from clinically approved medicines. bioRxiv. 2020. Publisher Full Text\n\nStoermer MJ: Homology models of Wuhan coronavirus 3CLpro protease. ChemRxiv. 2020. Publisher Full Text\n\nGlobal Health Drug Discovery Institute: Targeting 2019-nCoV Portal. 2020. Reference Source\n\nBeck BR, Shin B, Choi Y, et al.: Predicting commercially available antiviral drugs that may act on the novel coronavirus (2019-nCoV), Wuhan, China through a drug-target interaction deep learning model. bioRxiv. 2020. Publisher Full Text\n\nGao K, Nguyen DD, Wang R, et al.: Machine intelligence design of 2019-nCoV drugs. bioRxiv. 2020. Publisher Full Text\n\nLi Y, Zhang J, Wang N, et al.: Therapeutic drugs targeting 2019-nCoV main protease by high-throughput screening. bioRxiv. 2020. Publisher Full Text"
}
|
[
{
"id": "60688",
"date": "06 Mar 2020",
"name": "Qiaozhu Tan",
"expertise": [
"Reviewer Expertise Molecular Dynamics Simulation",
"Computer-aided Drug Design"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYu Wai Chen and co-workers presented a molecular modeling and docking study of the 3CL protease in the SARS-CoV-2 virus. The manuscript started with comparing polyprotein PP1AB sequences of SARS-CoV-2 and SARS-CoV, based on which the 3D structure of SARS-CoV-2 3CLPro protein was constructed. The authors then performed virtual screening against SARS-CoV-2 3CLPro using a library of 7173 purchasable drugs. Considering both binding affinities and known side effects, the authors recommend velpatasvir and ledipasvir, and further suggest combining them with another HCV RdRp inhibitor sofosbuvir, aka repurposing the Epclusa and Harvoni for treating the coronavirus. This is a concise and timely report, and has proposed new therapeutic possibilities for the SARS-CoV-2 virus. The manuscript could be further improved by addressing the following comments.\nMore details of the docking should be provided. What's the binding energy cutoff used? How is the hits (reported in Table 3) used? 3CLpro is catalytically active as a dimer. How is this considered in the virtual screening? What does the \"(B Top scorers)\" mean?\n\nIn the extended data of virtual screening, one compound could have multiple entries with different ZINC numbers. For example hesperidin corresponds to at least 20 different compounds. What are the difference? And how are different results assembled?\n\nTable 1 is not clear. Please do a column-by-column comparison between different sites of SARS-CoV and SARS-CoV-2. Also please add one-letter amino acid codes for the residues.\n\nThe constructed protein structure is very similar to the recently solved crystal structure (6LU7), as \"... confirms that the predicted model is good within experimental errors\", but the docking results seem to differ significantly. Could the authors explain?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Yes",
"responses": [
{
"c_id": "5377",
"date": "09 Apr 2020",
"name": "Yu Wai Chen",
"role": "Author Response",
"response": "1. In reporting our virtual screening results, we listed the top 10 scorers of each model. This is now added to Methods:VS. The ‘hits’ is the number of times a compound appears (representing different stereoisomers) out of the total number of possible stereoisomers of that compound in the library. For example, hesperidin has a large number of stereoisomers (38) in the library and 8 of these are among the top scorers, it can give some confidence of reproducibility of the screening method. Stereoisomers are expected to have different binding energies. This is clarified now in the Table 3 caption. The functional SARS-CoV 3CLpro is a dimer. Therefore the SARS-CoV-2 enzyme was also constructed as a dimeric model, preserving all intermolecular interactions. The A- and B-chain active sites were screened independently but each was derived from the catalytically-active dimeric model. This information was now included in the Methods: Preparation of structural model and the VS sections. The “(B Top scorers)” in the A column of Table 3 refers to those compounds which are top scorers of the B site but did not turn up among the A site top 10; they were listed to show that they still scored high (compared to the mean) although not at the top. This was explained in the Table caption. 2. The drugs-lib has 7173 purchasable drugs, with 4,574 unique compounds and their stereoisomers. Each entry is identified with the name of the compound as well as a ZINC15 ID. Stereoisomers of a compound (with same drug name but unique ZINC15 IDs) are collected together and presented as hits out of total possible stereoisomers. These are now described in Methods: VS and the Table 3 caption. 3. The purpose of Table 1 is to show that the variant positions do not overlap with any of the functional residues. It is not meant to be a detailed comparison. We amended the table to include one-letter residue identities. The list of dimerisation residues was revised to include R4, M6, E166 and E290. One reference was added. 4. The crystal structure virtual screening results (Table S2) is consistent with those of the models. Of the 9 top scorers, 6 were in common with the model top scorers (top half of table: Top scorers). For the remaining 10 of the model candidates, all of them were found in the full-list results of the crystal structure screen (bottom half of table: A, B model scorers), all with higher or similar scores to the two reference compounds, lopinavir and ritonavir. Given the semi-quantitative nature of VS and binding energy scoring using a static active site, these results from slightly different active-site conformations are considered to be agreeable."
}
]
},
{
"id": "60414",
"date": "06 Mar 2020",
"name": "Wai-Lung Ng",
"expertise": [
"Reviewer Expertise Medicinal Chemistry",
"Drug Discovery",
"Chemical 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\nSince December 2019, a plethora of cases resembling viral pneumonia has emerged exponentially in Wuhan, China, which is now coined Coronavirus Disease 2019 (COVID-19; formerly known as 2019-nCoV).\nChen et. al. reported the computational modelling and virtual screening results of the 3C-like protease (3CLpro) of SARS-CoV-2. This study is timely in view of the recent outbreak of COVID-19. The rationale of repurposing existing drugs to tackle the global viral outbreak is sound. The manuscript is also well-written and structured. It should be noted that:\nThe authors compared their model with the recently published crystal structure of 3CLpro and found a high similarity between the two structures. They also obtained a similar list of top-ranked drug candidates when the crystal structure was subjected to the same screening protocol.\n\nSeveral studies using similar modeling and virtual screening approaches have also been published recently.\n\nSome suggestions for improving the manuscript:\nThe authors proposed that the HCV drugs velpatasvir and ledipasvir, and thus Epclusa and Harvoni, could be attractive drug candidates for treating SARS-CoV-2 infection. However, there is no direct evidence to support this claim. To support this claim, the authors should connect the computational results with experimental data. To test their hypothesis, the authors should at least prove (or disprove) that the two HCV drugs could inhibit the biochemical activity of 3CLpro of SARS-CoV-2.\n\nTo further test the hypothesis, the two NS5A inhibitors should be tested using in vitro assays such as viral RNA PCR assay.\n\nIf there are no such experimental data to support the claim, the authors may consider revising their conclusion to \"the computational results provide a rationale for further experimental validation of treating SARS-CoV-2 with velpatasvir and ledipasvir\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5378",
"date": "09 Apr 2020",
"name": "Yu Wai Chen",
"role": "Author Response",
"response": "1. Experimental work to test these results is underway. This future plan is now included in the last sentence of the section Results:Assessment of the candidate drugs. 2. We agree that it is important to also study the effects of the two NS5A inhibitors. We plan to set up collaborations for that purpose. 3. This is related to point number 1. A sentence was added at the end of the section Results:Assessment of the candidate drugs to this effect."
}
]
},
{
"id": "60415",
"date": "17 Mar 2020",
"name": "Vip Viprakasit",
"expertise": [
"Reviewer Expertise Molecular genetics",
"Clinical haematology",
"Investigational New Drug",
"Clinical Trial."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Chen YW and his colleagues carried out virtual screening using the computational molecular modeling of the viral protein from SARS-CoV-2 or COVID-19 to identify therapeutics targets. The authors presented that 3CLpro protease enzyme of the SARS-CoV-2 is considered to be a promising drug target and repurposing accessible drugs to challenge the globally outbreaking of the SARS-CoV-2. The authors initially used the translated polyprotein (PP1AB) sequence of SARS-CoV-2 and SARS-CoV to prepare for protein structural model. Subsequently, they performed computational virtual screening against this against its using a library of purchasable drugs with the binding site grid. Among > 7,000 repurposing drugs in the screening with their known side effects, the antivirals ledipasir or velpatasvir are potentially used against SARS-CoV-2 infection with minimal side effects. The manuscript is straightforward in both terminology and structure. The manuscript can be considered to be accepted with a minor revision and could be further improved with following points:\nTable 2 is mentioned prior Table 1 in the manuscript.\n\nMore details of the setting and cut-off used in the virtual screening and analysis should be provided in the Method section.\n\nTable 1 is quite confusing. The importance residues of the SARS-CoV 3CLpro previously reported and the variant residues found in SARS-CoV-2 (this work) should be separated. The amino acid variants of each position should be included. Using the image with the annotation could be an alternative and more informative presentation.\n\nIn Table 3, some information should be included in the table, such as molecular weight. In addition, the authors should discuss more about the results shown in the Table 3 to compare the binding energy different between A and B chains.\n\nIn the conclusion, the authors proposed velpatasvir and ledipasvir as an attractive candidate. However, based on the virtual screening on the active sites of SARS-CoV-2 3CLpro model, both of them are not ranked as the top list in Chain A screening. Could you please explain this scenario? The results from other virtual screening package (such as Glide or FlexX) should be compared?\n\nTo extend the interest of the topic as well as to compare the potential for using repurposing drug in COVID-19 treatment, the drug virtual screening with other viral enzymes might be performed and compared. In this case, since there are several clinical researches for using this drug family (e.g. Lopinavir/ritonavir) in COVID-19 treatment, therefore, the authors can compare the virtual screening model with the clinical outcomes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Yes",
"responses": [
{
"c_id": "5379",
"date": "09 Apr 2020",
"name": "Yu Wai Chen",
"role": "Author Response",
"response": "1. This is now corrected. 2. In reporting our virtual screening results, we listed the top 10 scorers of each model. We employed a ‘soft’ cutoff at which all results having the same binding energy were included. This led to 10 (one non-human vet drug later excluded) and 11 results in chains A and B, respectively. This is now added to Methods:VS. We revised the Methods:VS section to make the procedures in MTiOpenScreen clearer.3. Table 1 is revised so that important residues in SARS-CoV and variant residues in SARS-CoV-2 are now separated. The amino acids are identified with their single-letter codes.We keep the two lists together because the reader can immediately compare them and see that the two sets do not overlap. The same information is conveyed in 3D on Fig. S1 in Extended data.4. Molecular weights were added accordingly. The ‘hits’ column was revised to represent the number of stereoisomers found, out of the total possible numbers. Table 3 caption was also revised to explain in detail.The two chains are structurally non-identical (rmsd in Cα=0.83Å) in the dimer and in the crystal structure. The differences lie primarily in side-chain conformations, especially for residues with long side chains. Thus, the two chains are two slightly-different conformational states of the protein. It is advantageous to have two states for VS because it allows some degrees of conformational variability of the active sites to be taken into account. AutoDock Vina implemented in MTiOpenScreen does not allow for the flexibility of the active site. Therefore, one would not expect the active sites on the two chains to yield the same results in terms of binding energies and ligand ranking. Further, the AutoDock Vina 'binding energy' is not a true binding free energy per se but an analogous empirical scoring function for the sake of assessment. We interpreted these results semi-quantitatively to extract trends, in VS results section, instead of exercising detailed energetic comparisons. 5. In VS results, we identified 16 candidates. Our first intention was to present all these to the readers as top scorers of docking ranked by AutoDock Vina binding energies. Next, in the Assessment section, we discussed the known properties of some of these which stood out, also considering their side effects. We highlighted velpatasvir and ledipasvir mainly because of their minimal side effects, which is a crucial factor in repurposing. In addition, we noticed the two drugs which correspond to these two compounds also contain sofosbuvir which was identified as an anti-SARS-CoV-2 candidate in a separate study. It was because of all these factors, velpatasvir and ledipasvir stood out among the 16 candidates. Therefore we highlighted them in the conclusion. We also mentioned other top-ranking candidates but they may have strong side effects. We briefly assessed these candidates to inform the clinicians who may be interested in these results. We did not intend for a comprehensive study comparing with other VS packages. Here we employed AutoDock Vina, one of the most popular VS utilities, and produced results for follow-up development. We are moving quickly to the next stage of experimental verification in order to respond to the rapidly worsening global crisis.6. This work was intended to be a fast response to the current pandemic — thus, we focused on one of the several potential targets for antiviral development. To this day, there are several parallel efforts on this and other targets at different scales. At the time we revised it (end of March, 2020), this article is outdated in some aspects. Therefore, we shall not extend the work further on the computational methods, as this is not the most pressing. As the reviewer mentioned, lopinavir and ritonavir have been clinically tried. These two compounds are also expected to be specific inhibitors of 3CLpro. Therefore, we checked to see if VS picked up these two drugs: indeed it did, with a medium score (now included in Table 3) quite a bit lower than the top candidates. Docking of lopinavir/ritonavir into the SARS-CoV 3CLpro has been performed previously (referenced in the main text) and our models have basically the same active sites. Disappointedly, the latest trial of lopinavir/ritonavir on COVID-19 showed no clinical benefit. We added a sentence in Discussion to update the readers about this clinical trial."
}
]
}
] | 1
|
https://f1000research.com/articles/9-129
|
https://f1000research.com/articles/9-248/v1
|
09 Apr 20
|
{
"type": "Research Article",
"title": "Implementation of social protection schemes for people living with HIV in three districts of Rajasthan state, India – a mixed methods study",
"authors": [
"B Gowthamghosh",
"Rosenara Huidrom",
"Visvanathan Arumugam",
"Neeraj Pathak",
"Neetu Purohit",
"Hemant Deepak Shewade",
"Anoop Khanna",
"Poonam Ramesh Naik",
"Rosenara Huidrom",
"Visvanathan Arumugam",
"Neeraj Pathak",
"Neetu Purohit",
"Hemant Deepak Shewade",
"Anoop Khanna",
"Poonam Ramesh Naik"
],
"abstract": "Background: In India, public social protection schemes for marginalized populations are extended to people living with HIV (PLHIV) and their households. Care and Support Centres (CSCs) linked to antiretroviral therapy (ART) centres play a key role in linking PLHIV to the schemes. In three districts of Rajasthan, India, we assessed the linkage of PLHIV registered at CSCs (2016–18) to social protection schemes and explored PLHIV and provider perspectives into barriers and suggested solutions for improving linkage. Methods: This was an explanatory mixed method study involving a descriptive quantitative phase using secondary data, followed by a descriptive qualitative phase involving face-to-face in-depth (five PLHIV) and key informant (three CSC staff) interviews. Results: Of 1123 registered PLHIV at CSCs, 1026 (91%) expressed willingness to avail social protection schemes. Of 1026, 94% were linked to any one scheme; 52% to Palanhar, 51% to bus fare concession and 42% to widow pension schemes. The perceived barriers were: authenticating Aadhaar number (unique identification number provided to all Indians using their registered mobile phone number), cumbersome application processes for each scheme, limited utility of travel concession, delayed and/or irregular disbursement of benefits and non-availability of comprehensive information, education and communication material with details of all schemes and their application processes. Reaching out to all PLHIV in the designated district was a huge task for outreach workers. Another important barrier was the potential disclosure of positive status to various stakeholders in order to avail the schemes. Suggested solutions were a single window default application process at ART centres with a smart card and a single pamphlet at ART centres with all the required details. Conclusions: Bus fare concession, Palanhar and widow pension schemes were the most widely accepted and linked schemes by PLHIV. Implementation barriers were identified that need to be addressed to improve the linkage.",
"keywords": [
"Social support schemes",
"utilization",
"HIV patients",
"social support",
"operational research",
"SORT IT"
],
"content": "Introduction\n\nGlobally, 36.9 million people are living with HIV (PLHIV). New HIV infections reduced from 3.4 million in 1996 to 1.8 million in 20171. HIV/AIDS can push people and households into poverty by reducing household labour capacity and increasing medical expenses. The average household income is lowered by 34% in households with a PLHIV when compared to households without a PLHIV2. Households with PLHIV are thus more vulnerable to suffer socioeconomic shocks. HIV-related stigma and discrimination further marginalize the households affected by the virus and exclude them from essential services2.\n\nWhile free and decentralized HIV services remove important barriers to access, other economic barriers like increased expenses for nutritious food and transport expenses, wages lost due to visits for antiretroviral therapy (ART) and social barriers, stigma and discrimination may exist3–5. Social protection helps PLHIV in overcoming these barriers5. According to the UNAIDS national composite policy index (2017), 109 countries stated that they had an approved social protection strategy, policy or framework; 99 were implementing various social protection schemes, and 85 countries’ strategies were HIV-sensitive at least to some extent6. Social protection refers to “all public and private initiatives that provide income or consumption transfers to the poor, protect the vulnerable against livelihood risks and enhance the social status and rights of the marginalized; with the overall objective of reducing the economic and social vulnerability of poor, vulnerable and marginalized groups”7.\n\nThere are around 2.1 million PLHIV in India. The significant impact of the national AIDS control programme (NACP) has been highlighted by a decline of more than 50% in the number of new annual HIV infections during the last decade8. Under NACP phase IV, social protection schemes are expected to play an important role in mainstreaming PLHIV9. Some schemes are for all vulnerable groups (HIV sensitive), while some schemes have been started specifically for PLHIV (HIV specific)10.\n\nChapter VII, Section 15.1, of the recent HIV and AIDS Prevention and Control Act 2017 guaranteed PLHIV access to welfare schemes11. In 2013, the review commissioned by the International Labour Organization on social protection for PLHIV identified 26 studies on access to social protection and its impact on people living with and affected by HIV, although no studies from India were included. A report by the United Nations Development Programme on HIV sensitive social protection in four Indian states aimed to understand the utilization, facilitating and constraining factors12. Since then, the social protection schemes under NACP-IV have been mainstreamed with a Care and Support Centre (CSC) linked to ART centres. The CSCs facilitate PLHIV upon ART registration to avail the social protection schemes. There is limited information from India about linkage to social protection schemes among PLHIV registered at ART centres and CSCs. Even though efforts are made to mainstream and provide various services to PLHIV, there is a huge gap in evidence on the reach and effectiveness of these services. Understanding the linkage to the social protection schemes quantitatively and qualitatively is crucial for planning appropriate interventions to improve the coverage of PLHIV availing the schemes.\n\nHence, we aimed to understand the linkage to key social protection schemes among PLHIV registered with CSCs and explore the barriers and suggested solutions to improve linkage from provider and PLHIV perspectives.\n\n\nMethods\n\nThis was an explanatory mixed methods study where the descriptive quantitative phase using secondary data was followed by a descriptive qualitative phase13. The theoretical framework underpinning the qualitative phase was content analysis14. In addition to the experiences and challenges faced by the PLHIV in availing the schemes, the qualitative phase explored the reasons for the findings of quantitative phase.\n\nThis study was conducted in three districts (Jaipur, Sikar, Churu) of Rajasthan, a state in north India during 2018–19. The estimated prevalence of HIV among general population in these districts is 0.75%, 0.0% and 0.5%, respectively. As of 2017, the total number of people on ART in the study districts was 37,0928. If a person is HIV-positive, post-test counselling is done and the person is referred to the nearest ART centre for clinical and immunological assessment and treatment. Treatment to PLHIV is provided through district level ART centres and all services are given free of cost.\n\nAll PLHIV are eligible to avail the state and/or central government provided (public) social protection schemes. There are seven widespread social protection schemes for PLHIV in Rajasthan and these schemes are extensions of schemes intended for vulnerable populations (Table 1). The CSCs support the health and wellbeing of all PLHIV and their affected families, with special effort to reach those from underserved populations, including women, children and members of high-risk groups, such as female sex workers, men who have sex with men, transgender people and people who inject drugs. The ‘Vihaan’ project by India HIV/AIDS Alliance is a national initiative establishing and managing 350 CSCs across India, and it manages the CSCs in the three study districts (there are a total of 17 CSCs in Rajasthan). Working in coordination with nearby ART centres, CSCs serve as safe spaces for PLHIV offering services that include counselling, outreach and follow-up support, health referrals, and linkages to social welfare schemes15.\n\nPLHIV, person/people living with HIV; ART, antiretroviral therapy; BPL, below poverty line.\n\n*Aadhaar card is a unique identification card used for various government purposes.\n\n** Government of Rajasthan started this scheme to transfer financial and non-financial benefits of governmental schemes directly to recipients in a transparent way. The Bamashah card and number is made for a family with a female member as the head of the family.\n\nPLHIV are counselled at the time of registration at the ART centre about the government sponsored welfare schemes and referred to the Vihaan-CSC. If the PLHIV is willing to avail any scheme, he/she will give consent to the in-charge of the Vihaan-CSC to process his/her application form on their behalf and becomes registered at the CSC. This process is voluntary. The PLHIV are given all details about the process of accessing the schemes and also informed about necessary supporting documents required (Figure 1).\n\nPLHIV, people living with HIV; SPS, social protection schemes; CSC, Care and Support Centre; ARTC, antiretroviral treatment centre.\n\nData related to key variables at registration and details on linkage to various schemes is routinely recorded in a paper-based register and single-entered into an MS Excel database at Vihaan CSC. Aggregate data is also available on a centralized Vihaan server.\n\nFor the quantitative phase, the study population was all PLHIV on ART who registered at CSCs between January 2016 and December 2018 from the study districts. For the qualitative phase, from the January to June 2018 (six months) cohort, PLHIV receiving benefits and not receiving benefits were purposively selected. To understand the experiences from the provider perspective, we included the following service providers (key informants) as they were routinely involved in contacting the PLHIV and linking them to social protection schemes: Vihaan staff at state and CSC level and outreach workers. The study participants were contacted over the phone and there were no refusals. The enrolment was stopped once saturation of findings was reached.\n\nFor the quantitative phase, the source of data was electronic records maintained by Vihaan centrally. Baseline socio-demographic (age, gender, education, occupation, income, marital status, number of PLHIV in family) and clinical data (CD4 cell count, alcohol use, tobacco use, risk behaviour) were extracted from the CSC registers. The linkage to the various social protection schemes was assessed as of April 2019. Data analysis was carried out in June 2019.\n\nFor the qualitative phase, face-to-face interviews were conducted (June–August 2019) to understand PLHIV (in depth interviews) and provider (key informants) perspectives into the barriers and suggested solutions for improving linkage to schemes. The principal investigator (BG) is a public health professional (M.P.H.) who is trained in qualitative research methods. He conducted the face-to-face interviews in the local language (Hindi) after obtaining their written informed consent to participate in the study. Interviews were conducted at the CSC office in a private room. The principal investigator, though aware of ART services and social protection schemes, was not involved in routine programme implementation and belonged to a public health academic and research institute (external expert). A qualitative interview guide was pilot tested on three PLHIVs from the CSC office, who were not part of the final sample. A few changes were made after the pilot testing; probes were added and local terms used in the final guide. The final interview guide comprised of broad open-ended questions and is available as Extended data16. The interview guide was further modified based on the quantitative phase results. Only the study participant and the principle investigator were present during the interview. Audio recording (after consent) and/or verbatim notes were taken during the interview. After the interview was over, the summary of the interview was read back to the participants to ensure participant validation.\n\nThe electronic data obtained from the Vihaan-CSC were cleaned and exported to SPSS (version 21 IBM SPSS) for analysis. Continuous variables were summarized in terms of either mean ± SD or median (IQR) depending on the statistical distribution of data. Categorical variables were summarized using frequencies and proportions.\n\nFor face-to-face interviews, transcription and translation was done the same day based on the verbatim notes and audio recordings. Manual descriptive thematic analysis was done by two investigators (BG and HDS)13,17. The decision on coding rules and theme generation was done using standard procedures and in consensus18. The themes were derived from the data. The codes/themes were related back to the original data19.\n\nThemes/categories have been reported below in single quotation marks, verbatim quotes in double quotation marks and italicized, author explanation within quotes in square brackets and respondents' details in round brackets. The findings were reported by using ‘Consolidated Criteria for Reporting Qualitative Research’ (COREQ) guidelines20.\n\nEthics approval was obtained from the Institutional Ethics Committee of the IIHMR University, Jaipur, India (May 2019/2) and the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease (The Union), Paris, France (No 127/18). As the quantitative phase of the study involved secondary data, a waiver for informed consent was sought and approved by the ethics committees. For the qualitative phase, written informed consent was obtained for participation and publication of findings and this process was approved by the ethics committees. Throughout the study, efforts were made to avoid inadvertent disclosure of participants’ HIV status with others.\n\n\nResults\n\nBaseline characteristics. The sociodemographic and clinical characteristics of 1123 PLHIV registered at the CSCs, stratified by study districts, are shown in Table 2 and Table 3. The mean age was 34 (±12.8) years and 46% were female. Of 1123 registered at CSCs, 63.9% had a monthly household income below 70 USD and 53% (n=604) PLHIV had one more person with HIV in the household. At CSC registration, 467 (41.6%) had CD4 count <350 cells/mm3 (median count 364 cell/mm3), 132 (12.0%) consumed alcohol and 25% had daily usage of tobacco (smoking or chewing). Regarding the possible source of HIV transmission, in 478 (42.6%) it was reported to be heterosexual sexual transmission and in 359 (32.0%) it was reported as unknown.\n\nCSC, Care and Support Centre; PLHIV, people living with HIV; ART, antiretroviral therapy.\n\n*Number other than the registered.\n\nPLHIV, people living with HIV; CSC, Care and Support Centre.\n\nLinkage to social protection schemes. The linkage to the social protection schemes is presented in Table 4. Of 1123 PLHIV registered at CSC, 1026 (91.3%) expressed willingness to be linked to schemes and of them 93.6% (n=961) were linked to at least one scheme; 51.8% (n=498) to Palanhar (cash assistance for children of PLHIV), 50.7% (n=488) to bus fare concession, 14.8% (n=143) to Antyodaya Ann Yojana (meaning scheme to provide subsidized food to the socio-economically weakest) and 41.8% (n=54) to widow pension (among 129 widows living with HIV). Overall, linkage to the social protection schemes is good, but the individual scheme linkage varies among the districts. In the case of bus fare concession, the linkage is good in Sikar and Churu, but in Jaipur it is very poor. Linkage to Palanhar and widow pension schemes are good in Jaipur but poor in the other two districts. The variation in the linkages to the schemes prompted questions about the barriers in linking the schemes at a PLHIV and CSC level.\n\nPLHIV, person living with HIV; CSC: Care and Support Centre; AAY, Antyodaya Ann Yojana.\n\n^ Individual patient data not available for Mahatma Gandhi National Rural Employment Guarantee Scheme, Bamashah and Rail concession were excluded.\n\n+Current marital status as widow.\n\nTable 5 depicts the participant characteristics and duration of each one-to-one interview. In the context of linking and accessing the social protection schemes, we present the results in two parts: perceived barriers and suggested solutions by the PLHIV and program staff.\n\nPLHIV, people living with HIV; CSC, Care and Support Centre; SPS, social protection schemes.\n\nPerceived barriers - PLHIV perspective. Thematic analysis showing barriers in availing the social protection schemes from the perspective of PLHIV are depicted in Figure 2. We identified five themes, broadly grouped into two categories: system level and patient level.\n\nPLHIV, people living with HIV; CSC, Care and Support Centre; IEC, information education and communication.\n\nSystem level\n\nFour themes were categorized here. ‘Linking Aadhaar with bank and cumbersome application process’ was consistent among all participants. The common issue was in authenticating the Aadhaar number [unique identification number provided to all Indians that is linked to a bank account] through a one-time password sent to the registered mobile number of PLHIV. The Aadhaar number facilitates the direct benefit transfer of schemes like the widow pension and Palanhar scheme. If the PLHIV changes his or her registered mobile number, then this authentication is delayed. One participant mentioned about the inconsistency of basic information in other government documents (like name from school records did not match with Aadhaar) causing delay in scheme linkage. As the bank account information needed the child’s name, the name mismatch in records resulted in delay.\n\n“For Aadhaar update my mobile number registered with Aadhaar got lost, I have updated my new mobile number and received message but I don’t understand the message [message with one-time password details which has to be shared with the concerned department]\" (Male PLHIV)\n\n\"The child’s name in school record varies with the name recorded in Bamashah document [Government of Rajasthan started this scheme to transfer financial and non-financial benefits of governmental schemes directly to recipients in a transparent way. The Bamashah card and number is made for a family with female member as the head of the family]. I have updated the name in Bamashah. \". (Male PLHIV)\n\n‘Limited utility of travel concession’ was observed. The concession for the bus fare was limited to one trip per month. For an additional trip in the same month, the PLHIV had to pay the full fare. ‘Delayed receipt of benefit due to budget delay’ was a common observation.\n\n\" I have submitted the form for Palanhar scheme nearly 12 months ago, but due to lack of budget, I am still waiting to receive the money\". (Female PLHIV)\n\n‘Non-availability of comprehensive IEC material’ was noted. One of the important elements in accessing the schemes is the availability of the information, education and communication (IEC) material. However, there was no comprehensive IEC material available from the government and most of the PLHIV did not have adequate knowledge of all schemes. The only printed handout available was from the Vihaan containing the list of schemes and required documents.\n\n\"I know Palanhar Yojna [scheme] only. [That too,] Once [after] I heard from the other patients about this scheme\". (Female PLHIV)\n\nPatient level\n\n‘Lack of family support’ in linking to the schemes is a barrier due to stigma and discrimination at the family and community level. Apart from the system level support, the PLHIV need significant support from the family in overcoming the economic burden caused due to loss of employment and social stigma.\n\n\"Nobody is there to look after the children. My mind is not working. No assistance at home\". (Male PLHIV)\n\nPerceived barriers - provider perspective. Thematic analysis showing barriers in availing the social protection schemes from the providers’ perspective is depicted in Figure 3. We identified ten themes, broadly grouped into two categories: system level and patient level.\n\nPLHIV, people living with HIV; CSC, Care and Support Centre; ART, antiretroviral therapy; IEC, information education and communication.\n\nSystem level\n\nAs expressed by the PLHIV, the program staff also expressed the same barriers related to - ‘linking Aadhaar with bank and cumbersome application process’, ‘limited utility of travel concession’, ‘delayed/irregular disbursement of benefit’ and ‘lack of availability of comprehensive IEC material’.\n\n‘Renewal’ of certain schemes [Palanhar, Bus fare concession] is essential for receiving continued benefits. The program staff reported that many PLHIV forgot to submit the renewal documents, after which the benefit was discontinued.\n\n\"Every year they [PLHIV] have to submit the school enrollment certificate through online, failing to which they [beneficiary] cannot receive the benefit\". (Male program staff)\n\nThe application process also required repeated visits to offices by PLHIVs after their application, leading to long periods of time between application and linkage. PLHIV and outreach workers had to make multiple visits to the office due to non-availability of the staff.\n\n\"The department has few staff to run the office so once the form is filled and submitted, we [program staff] have to visit again and again to the offices whether the scheme started or not.\" (Male outreach worker)\n\n\"Pension has a long lead time, it will take 3 to 4 months to get the pension\". (Male program staff)\n\nThe ART centres had single hoardings / posters summarizing all the schemes, but handouts that could be given to PLHIV were unavailable at ART centres. Reaching out to all the PLHIV in the designated district by the outreach worker was a huge task.\n\n\"We do not have much resources for mobilization, somehow this is one of the reasons of clients lost to follow-up\". (Female program staff)\n\nAnother important barrier was the potential ‘disclosure of positive status’. The PLHIV did not want to disclose their status due to fear of discrimination and social exclusion and preferred to travel to far away health facility, resulting in the burden of transportation expenses21,22. Though all the schemes applicable to below poverty line families have been extended to PLHIV, in many times the status had to be disclosed to avail the scheme.\n\n\"PLHIV has to appeal to SDO [sub-divisional officer] for Khadya Suraksha Yojana [Food security scheme], SDO asks why they want to avail the schemes and they have to disclose the HIV status\". (Male program staff)\n\n\"Collector [ District administrator] also ordered that other than the gram sachiv [village government official] the PLHIV need not disclose their HIV status to anyone else, because the sachiv [secretary of local administrative body] is appointed by the government and the sarpanch [elected village representative] is a political post. Sachiv will think about disclosing the status before telling others, he cannot risk his job\". (Male program staff)\n\n‘Reluctant doctors’, as described by the program staff, discriminated against the PLHIV and this is a major barrier in accessing health care services. Evidence suggests that the PLHIV are prone to being discriminated against by the health workers at health facilities23–26.\n\n\"Big challenge is with the government hospital. The doctors are not operating the PLHIV after knowing the HIV status\". (Male program staff)\n\nPatient level\n\nThe program staff perceived that ‘non- disclosure of HIV status to spouse causing in delay of treatment27,28, delay in submitting the renewal documents’ for schemes like Palanhar that need annual renewal and ‘irregular visits to ART centre’ due to transport charges were barriers in linking the schemes.\n\n\"We have many instances reported in which the wife is unaware of the husband’s HIV status\". (Female program staff)\n\n\"Irregularity in applying for renewal for the scheme, may be due to community ignorance\" (Male program staff)\n\nSuggested solutions. The suggested solutions are described as two themes from the PLHIV perspective (Table 6) and seven themes from provider perspective (Table 7).\n\nPLHIV, people living with HIV; CSC, Care and Support Centre.\n\nPLHIV, people living with HIV; CSC, Care and Support Centre; ART, antiretroviral therapy; IEC, information education and communication; NGO, non-governmental organization; PHC, primary health centre.\n\nDocumentation\n\nThe PLHIV suggested a simpler process for scheme application and the providers suggested a centralized process at ART centre. A procedure change was suggested by the providers in which a unique smart card could be issued at ART centre. This smart card should be linked with Aadhaar or a bank account and used for availing all the schemes. Special camps for PLHIV run by the government to redress the issues and barriers in linking and availing the schemes by the PLHIV were other suggested solutions.\n\nAccessibility\n\nIn the process of continuous and uninterrupted disbursement of scheme benefits, adequate budget allocation is necessary. Awareness generation among the PLHIV, officials and at the community level about the schemes through strong comprehensive IEC material [all schemes’ related information in single pamphlet] should be made available widely. In addition, a resource book on social protection schemes was also recommended.\n\n\nDiscussion\n\nThis is one of the few mixed methods studies on this topic conducted in India. All PLHIV are eligible to avail the state and/or central government provided social protection schemes. These schemes are extensions of schemes intended for marginalized populations. Of all other schemes available in Rajasthan, bus fare concession, pension and the Palanhar scheme were the most widely accepted and linked schemes by PLHIV. Even with certain limitations like limited travel (one trip in a month using bus fare concession), long period of time before the receipt of benefits (2–3 months for pension) and complex documentation process (Palanhar), PLHIV are considering these schemes.\n\nVerification of Aadhaar and the complicated application process for linking to the schemes and renewal were the key barriers. A PLHIV has to submit 7-9 different supporting documents for availing the schemes over repeated visits, and delayed benefit and the need for renewal of certain schemes are causing distress to PLHIV. Both PLHIV and the program staff reported that the process is not user friendly.\n\nThe ART centre is the first point of information on social protection schemes and CSCs play a key role in linking to the schemes. Very limited comprehensive IEC material related to social protection schemes was available at the ART centres and CSCs.\n\nOne of the key areas of the CSCs is community mobilization. Due to lack of adequate funds and human resources, the program staff (CSC) were not able to visit all the PLHIV at village level in a timely manner. The care and support centre has done an impressive job in linking the social protection schemes to the PLHIV who consented to them; however, since the ART centre is the first point of contact and also a sustained point of interaction, it is felt that ART centre must play a lead role in facilitating the linkage of social protection schemes.\n\nThe investigators recommend the following. First, a single window default application process at ART centres could be established, providing PLHIV with a smart card. The registration at this window can enable the PLHIV to access multiple schemes as per their eligibility with the help of smart card. This window can help the PLHIV in choosing and applying to the schemes with ART registration as a basic requirement.\n\nSecond, a state or national level direct benefit transfer (DBT) scheme for PLHIV upon registering at the ART centre could be considered, like Nikshay Poshan Yojana, where all the notified tuberculosis patients under treatment are eligible for incentives29,30. The DBT may be in the form of conditional or unconditional transfers (cash/food). Evidence from South Africa31,32 revealed that the HIV-risk has been reduced among adolescent girls and boys with the model of “cash plus care” (cash transfers with psychological support). A cross-sectional study in Cameroon found that financial barriers decrease ART adherence33. Household economic strengthening interventions can help the PLHIV in meeting the direct non-medical and indirect costs (loss of wages due to sickness absenteeism)34. Recently, initiatives of the state government for relaxation of the age criteria for the widow pension scheme to all HIV positive widows has been an important enabling factor for widows to avail the scheme.\n\nThere are some limitations of the study. We did not have data for PLHIV who did not register at the CSC; therefore, the true picture of linkage among PLHIV registered at ART centres may be even lower and we were not able to make observations on that aspect. Delays in linkage were not captured quantitatively. We did not interview the key stakeholders (the government departments) who are responsible for delivering the schemes.\n\nMore studies need to be conducted on the impact of linkage to social protection schemes on the lives of PLHIV. A pilot intervention to test the suggested solutions and the impact on linkages to the schemes is recommended.\n\n\nConclusions\n\nThe study was able to identify the barriers perceived by the PLHIV and providers. CSCs have been by far the major facilitators in linking the schemes. The ART centres did not seem to play a significant role in facilitating the social protection schemes, except providing ART. Comprehensive IEC need to be developed and widely circulated at all levels of service delivery. There is an urgent need to address these barriers to enhance linkage to social protection schemes.\n\n\nData availability\n\nThe quantitative and qualitative data that support the findings of this study can be obtained from India HIV/AIDS Alliance. These data are not publicly available due to concerns that the data could potentially disclose individuals’ identities. Data are, however, available upon request to researchers for the purpose of further academic research from the corresponding author (ghoshbg@gmail.com) and with permission of India HIV/AIDS Alliance.\n\nFigshare: Annex 1.pdf. https://doi.org/10.6084/m9.figshare.11889630.v116.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank the participants who kindly agreed to participate in the study and share their experiences. We are also grateful to have received the encouragement and support by the IIHMR University, Jaipur management.\n\nDisclaimer: The contents of this paper do not necessarily reflect the views of the Government or Non-Governmental Organizations or The Union\n\n\nReferences\n\nUNAIDS: Miles to go: Closing Gaps Breaking Barriers Righting Injustices. 2018 [cited 2018 Nov 15]. Reference Source\n\nNACO, Vrutti, UNDP: Reducing vulnerabilities: Key social protection schemes from a PLHIV perspective. 2011 [cited 2018 Nov 16]. Reference Source\n\nUNICEF, UNAIDS, IDS: Enhancing Social Protection for HIV Prevention, Treatment, Care & Support - The State of the Evidence. [cited 2018 Nov 15]. Reference Source\n\nKyaw NTT, Kumar AMV, Oo MM, et al.: Long-term outcomes of second-line antiretroviral treatment in an adult and adolescent cohort in Myanmar. 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Report No.: ISBN 92 4 159073 4. Reference Source\n\nNakigozi G, Atuyambe L, Kamya M, et al.: A qualitative study of barriers to enrollment into free HIV care: perspectives of never-in-care HIV-positive patients and providers in Rakai, Uganda. Biomed Res Int. 2013; 2013: 470245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Health and Family Welfare Government of India: Gazette Notification For DBT with NIKSHAY. D.O. No. Z-28015/24/2017-TB. 2017; 1–17.\n\nRevised national tuberculosis control programme (RNTCP); Central TB division; Ministry of Health and Family Welfare; Government of India: NISHAY - A web based solution for monitoring of TB patients.\n\nCluver LD, Orkin FM, Boyes ME, et al.: Cash plus care: social protection cumulatively mitigates HIV-risk behaviour among adolescents in South Africa. AIDS. 2014; 28(Suppl 3): S389–97. PubMed Abstract | Publisher Full Text\n\nRudgard WE, Carter DJ, Scuffell J, et al.: Cash transfers to enhance TB control: lessons from the HIV response. BMC Public Health. 2018; 18(1): 1052. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoyer S, Marcellin F, Ongolo-Zogo P, et al.: Financial barriers to HIV treatment in Yaoundé, Cameroon: first results of a national cross-sectional survey. Bull World Health Organ. 2009; 87(4): 279–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoret W, Carmichael J, Swann M, et al.: Household economic strengthening and the global fight against HIV. AIDS Care - Psychol Socio-Medical Asp AIDS/HIV. 2018; 30: 1–5. Publisher Full Text"
}
|
[
{
"id": "71736",
"date": "14 Oct 2020",
"name": "Mulamuli Mpofu",
"expertise": [
"Reviewer Expertise Public Health",
"HIV/AIDS",
"Malaria",
"Integrated Vector Management",
"Health Information Systems",
"Epidemiology"
],
"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\nArticle summary:\nThe study assessed access to social protection schemes by PLHIV in three districts of Rajasthan State in India. This was a mixed methods study integrating secondary data analysis to assess access rates of the different social protection schemes by district. The qualitative component through interviews (Vihaan staff, outreach workers and PLHIV) were conducted to understand the system and individual barriers to access to social protection schemes. The authors concluded that ART centers did not play an important role in facilitating access to social protection schemes.\n\nComments by section:\nAbstract:\nAbstract is consistent with the main body. As in the description of the qualitative phase; indicate what was assessed in the secondary data analysis.\n\nIntroduction:\nParagraph 4 - it will be important to list the type of schemes that PLHIV are entitled to per the HIV/AIDS Prevention Control Act.\n\nMethods:\nStudy design: This was a mixed methods study\n\nSetting - The HIV prevalence for Sikar is given as 0.0% and this brings to question where the PLHIV came from. Maybe increase the decimal places for the percentage if it is less than 0.1.\n\nTable 1: Provide description of the purpose of each SPS. Are these schemes exclusively for PLHIV or beyond? This is not coming out clearly.\n\nFigure 1: Everyone ends up accessing the SPS whether consented or not. Perhaps more details on those who did not consent so that its clear how they end up getting SPS.\n\nAnalysis - Only descriptive analyses were conducted but it would have been good to also assess for statistical difference in these measures by sex, district etc. This will help understand the observed trends.\n\nInterviews - its not clear how the questions were translated. Were they translated after transcription or during? Its also not clear how the individuals interviewed were selected. The authors also indicate that 'The enrolment was stopped once saturation of findings was reached\", its highly unlikely saturation can be reached by interviewing three or five individual using an open-ended tool.\n\nEthics section - how was the confidentiality of the interview participants, particularly the PLHIV protected? While the authors indicate that an NRD was obtained for the secondary analysis, it will be worthwhile indicating that: \"non of the authors interacted with the data that contained personal identifying information'.\n\nResults:\nThe results are presented as three separate studies - secondary analysis, qualitative study for PLHIV and qualitative study for providers. The purpose of a mixed-methods study is to triangulate the results where the qualitative explain the quantitative; and qualitative results from different sources get analyzed together. The authors should revisit the results section.\n\nDiscussion:\nThe discussion does not interpret and explain the findings. It does not integrate literature to make comparisons with other areas.\n\nConclusions:\nThe conclusions should be directly related to the results. There wasn't much analysis done from the quantitative data and the sample size from the interviews may not have sufficient power for such conclusions to be drawn.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "82651",
"date": "04 May 2021",
"name": "Georges Nguefack-Tsague",
"expertise": [
"Reviewer Expertise Statistics",
"Modeling",
"Health Informatics",
"Health Information System (HIS)",
"SGD monitoring and evaluation",
"public health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease update the number of people living with HIV (PLHIV) and also new infections in 2020 globally and in India.\n\nClearly justify the choice of the three districts.\n\nTable 3, justify the choice of CD4 count cutoffs; I would rather prefer that you use WHO or UNAIDS cutoffs.\n\nTables 2-4, please insert a column on p-values to statistically compare variables across districts, i.e. statistical significance. We understand that the quantitative phase was descriptive, but it would be great to consider significance.\n\nIn addition to sex, please add age to the Verbatim quote\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-248
|
https://f1000research.com/articles/9-247/v1
|
08 Apr 20
|
{
"type": "Research Article",
"title": "Phytochemical investigation and assessment of the hypoglycemic activity of two herbal extracts from selected Iraqi medicinal plants in alloxan-stimulated diabetic rats: a comparative study",
"authors": [
"Amjed Haseeb Khamees",
"Hayder Adnan Fawzi",
"Hayder B. Sahib",
"Amjed Haseeb Khamees",
"Hayder B. Sahib"
],
"abstract": "Background: Cyperus rotundus and Trigonella foenum graecum (fenugreek) have been utilized for many years as medical applications, mostly as traditional natural medications. The present research was designed to assess the hypoglycemic activity of the ethanol extract of Iraqi fenugreek seeds and tuberous roots of C. rotundus in alloxan-stimulated diabetic rats and to evaluate the diverse phytochemicals found in ethanol extracts that may have therapeutic activity. Methods: Qualitative assessment of diverse secondary metabolites by corresponding chemical tests was been used to specify each group of compounds in the crude extracts of both plants. Male Wistar rats (n=40) were subdivided randomly into four main groups: group A, alloxan-stimulated diabetic rats with administered with distilled water (10mL/kg; control); group B-D, alloxan-stimulated diabetic rats administered with ethanol extract of C. rotundus (10 mg/kg; group B), glibenclamide (10 mg/kg; group C), and ethanol extract of fenugreek seeds (15mg/kg; group D). These medications were given orally for 6 weeks. Blood glucose of the rats was measured at baseline and after 1, 2, 4, and 6 weeks after administration of medication. Results: Phytochemical analysis showed the presence of many secondary metabolites including alkaloids, flavonoids, anthraquinones, tannins, and steroids, in the crude extracts of C. rotundus and fenugreek seeds. After 6 weeks all groups of rats showed a significant decline in blood sugar level compared to the control group. Group C (glibenclamide) showed the lowest value, followed by group D (fenugreek seeds) and group B (C. rotundus). Conclusions: Ethanol extract of both plants possess considerable hypoglycemic activity in alloxan-stimulated diabetic rats, with superior efficacy shown by fenugreek seed extract, after 6 weeks of study.",
"keywords": [
"Trigonella foenum graecum",
"Cyperus rotundus",
"alloxan-stimulated diabetes rats",
"hyperglycemia effects"
],
"content": "Introduction\n\nDiabetes mellitus (DM) is a group of syndromes characterized by high blood sugar; modulated metabolism of lipids, carbohydrate and proteins; and an elevated risk from vascular diseases. It is associated with a metabolic defect with relative or absolute insulin deficiency and/or insulin resistance. Most patients can be classified clinically as either insulin-dependent DM or insulin-independent DM1. Chronic complications include cardiovascular disorders, central nervous system damage, chronic kidney diseases, complication of the retina, and difficulties in wounds healing2. These complications have a pronounced effect on the quality of life of the patients by increasing morbidity and mortality2. Due to the multifactorial pathological nature of DM, it requires several therapeutic agents for optimal management. Most research on DM focuses on early diagnosis and preventive measure in highly at risk people.\n\nTrigonella foenum graecum (fenugreek) has an extended range of applications (medical and non-medical), e.g. a flavoring agent of food3, use as a diuretic agent, ability to reduce blood pressure4 and blood cholesterol, cardiotonic and hypoglycemic properties, ability to enhance insulin production, antidiabetic effects, and antinociceptive and anti-inflammatory effects5. Fenugreek has been widely used as an ingredient of many blood glucose lowering substances, from different plant parts, including seeds and leaves and extracts in diverse solvents6. Its chemical component consists of different categories like saponins, sapogenins, alkaloids, and flavonoids. The hypoglycemic activity of fenugreek has been studied in different research; with the seeds of the plant as the main focus since its control over diabetes7. Cyperus rotundus (known as saed in Iraq) develops from a system of underground dark green tubers8. The tubers of the saed are utilized in many therapeutic indications, including as an antipyretic, anthelmintic, hypotensive, antihistaminic, and smooth muscle relaxant9,10.\n\nThe current work aimed to study the phytochemical properties, and the hypoglycemic effect of the ethanol extract of C. rotundus and fenugreek seeds in alloxan-stimulated diabetes rats. This is a preclinical study to assess these new compounds.\n\n\nMethods\n\nThe study conducted at the College of Pharmacy, Al-Nahrain University, Iraq, during the period from November 2019 to January 2020.\n\nAll procedures performed in the study were approved by the Institutional Research Committee at the College of Pharmacy, Al-Nahrain University (Ref# 2019/0078) and in accordance with the animals Act 1986 (ASPA) for animals used in scientific research.\n\nAll their efforts were made to reduce animal suffering by provision of water and food regularly, changing the beds every other day, letting animals sleep during the night offering a calming and dark place, and checking for any inconvenience to the animals throughout the study.\n\nA total of 40 Wistar male rats (175 – 230 g weight) were used in the study. The animals were obtained from animal house at the Iraqi National Drug Quality Control Center and Drug Research (Baghdad, Iraq). The animals were individually caged and allowed free access to water and standard rodent chow diet (18% Protein, 5% fat, Envigo Teklad Global Rat Food Pellets 2018), food was provided in a standard stainless-steel hopper. The animals were maintained at room temperature (23 – 25°C), 30–40% humidity, and lights on from 7 AM to 5 PM. The rats were maintained in polypropylene cages with husk as the bedding material. The animals were kept for 7 days after transfer from their original housekeeping to allow accommodation before performing the study.\n\nA resource equation approach was used (since it is not possible to assume the standard deviation and the effect size for this study) and the expected statistical analysis was ANOVA, based on the following equation:\n\nAnimals were injected intraperitoneally with a single dose of alloxan monohydrate (Sigma Aldrich, USA) (120 mg/kg) in saline (0.9% NaCl) to induce diabetes13. The rats were fasted overnight for 12-13 hr but allowed access to water before blood glucose level was measured. After 72 h, glucose levels were recorded using a glucometer. Rats with blood glucose levels of 200 mg/dL and above were considered as diabetic and were selected for the study14.\n\nThe alloxan-induced diabetic animals were subdivided into four groups using a randomized block design (see below): group A, group A, alloxan-stimulated diabetic rats with administered with distilled water (10mL/kg; control); group B–D, alloxan-stimulated diabetic rats administered with ethanol extract of C. rotundus (10 mg/kg; group B), glibenclamide (10 mg/kg; group C), and ethanol extract of fenugreek seeds (15mg/kg; group D) (Table 1).\n\nRandomized block design was selected as the experimental module. The rats were divided into four blocks: block one (group A) given distilled water at first week, block two (group B) started treatment the next week, block three (group C) started treatment the third week, and block four (group D) started treatment the 4th week. All the animals were kept in the same conditions. The previous order established when each block received their treatment, then in each block serum blood glucose was measure at baseline, 1 week, 2 weeks, 4 weeks, and 6 weeks after the initiation of treatment (no rat were sacrificed in the study). The allocation of rats was random in each block with the matching of between block in rats’ weight, age, diets, and other fixed parameters. The treatment was considered a fixed effect.\n\nEach of the groups contained 10 rats (total 40 rats).\n\nAll treatments were administered to the rats with 1.0 cc syringe using an oropharyngeal cannula. Doses were decided upon after review of the literature, taking into consideration of trying to provide the lowest dose possible.\n\nAssessment of the change in blood glucose from baseline and after 1 week, 2 weeks, 4 weeks, and 6 weeks after the administration of the treatment.\n\nThe glucose level in the blood was measured with a glucometer (ACCU-CHEK Active, Mannheim, Germany), and values > 200 mg/dL were considered diabetic15. The animals were kept on fasting status for 12 – 13 hrs on days of sampling, and blood samples were obtained from tail vein by tail nipping procedure (no anesthesia was required). Blood glucose was measured early in the morning after overnight fasting14.\n\nExperimental diabetes in rats was induced by injecting alloxan monohydrate (Sigma Aldrich, USA) at a single dose of 150 mg/kg, i.p. in ice-cold normal saline. After 72 h, glucose levels were recorded using a glucometer. Rats with blood glucose levels of 200 mg/dL and above were considered as diabetic and were selected for the study.\n\nThe Iraqi plants were collected from Baghdad, in November 2019 and the plants were specified and authenticated by the Biology Department, College of the Sciences, Baghdad University, Iraq. The rhizomes of C. rotundus, and seeds of T. foenum graecum were obtained.\n\nThe preparation of extract was performed using the method described by Ali et al16. In total, 500 g of coarse powdered C. rotundus (rhizomes were coarsely grounded and sieved) was loaded into a Soxhlet apparatus for the extraction process. The powder was first defatted with petroleum ether solvent (60–80°C) before extraction with ethanol. The ethanolic extract of 500 g T. foenum graecum seeds was obtained from the reflex apparatus that was prepared two days previously (defatted extract). The ethanol extracts were evaporated using a rotatory evaporator and dried in a water bath.\n\nChemical tests to assess active constituents in the plants were done using plant crude extracts and standard procedures17 (Table 2).\n\nReproduced with permission from 18.\n\nTwo-way ANOVA test was used to analyze the difference in blood glucose throughout the study. All analysis carried out using SPSS version 22.1 (Chicago, IL). P-value was considered to be significant if <0.05.\n\n\nResults\n\nThe phytochemical analysis revealed the presence of many secondary metabolites, including alkaloids, flavonoids, anthraquinones, tannins, and steroids (Table 3).\n\nAt baseline, there was no significant difference in blood glucose levels between rats as they had typical blood glucose levels (range, 73.4 ± 5.2 to 78.8 ± 5 mg/dl) at the beginning of the experiment.\n\nAt 1 week, group D (fenugreek seeds) showed a similar reduction in glucose levels to animals in group B (C. rotundus) (p-value = 0.99). This reduction was significantly more than group A (negative control) (p-value < 0.001).\n\nAt 2 weeks, there were no significant differences in values between groups D and B, and both groups showed no significant differences compared to group C (glibenclamide).\n\nAt 4 weeks, the results showed a significant decline in all groups' blood glucose readings from baseline. There was no significant difference between groups C and D.\n\nAt 6 weeks, the final readings showed that all groups had a significant decline in blood glucose level compared to the group A. Group C had the lowest value, followed by group D and group B. Figure 1 shows the assessment of glucose levels during the whole study period.\n\nGroup A, distilled water (10mL/kg); B, Cyperus rotundus ethanol extract (10mg/kg); Group C, glibenclamide (10mg/kg); Group D, fenugreek seeds ethanol extract (15mg/kg).\n\nNo adverse effects were reported during the study period.\n\n\nDiscussion\n\nThe existence of these groups of compounds provides scientific evidence for the previously presumed medical applications of C. rotundus in the treatment of specific disorders. The existence of flavonoids, alkaloids, tannins and terpenoids appears to be considerable. T. foenum graecum also showed positive results for alkaloids, anthraquinones, tannins, and steroids, while the extract exhibited negative results towards flavonoids.\n\nThe results indicate that both ethanol extracts of fenugreek seeds and C. rotundus reduced the blood glucose level in diabetic rats. This hypoglycemic effect shows that these extracts have medicinal activity. The bioactive constituents of both extracts may play a role in increasing insulin secretion from β-cells19. Other supposed mechanisms may include this insulin-like effect or other modes of action, such as initiation of peripheral tissue glucose uptake, inhibition of endogenous glucose output, or enhancement of gluconeogenesis process in corresponding tissues, for example the liver and muscle tissue. A similar mode of action has been shown for other plant extracts with hypoglycemic activity20. In addition, it is established that flavonoids control aldose reductase enzyme21. Many researchers report that there are several flavanols that can control diabetes, by bringing about restoration of pancreatic islet cells and increasing insulin production in streptozotocin-induced diabetes. In addition, it may also enhance Ca2+ uptake from isolated islet cells thus giving an indication that is very effective in type II DM22. Further analyses are needed to detect the complete mode of hypoglycemic action of these plants.\n\n\nConclusion\n\nThe current study observed that fenugreek seeds and C. rotundus ethanol extracts possess considerable hypoglycemic activity in alloxan-stimulated diabetic rats with considerable higher activity for fenugreek seeds in contrast to C. rotundus tuberous roots extract. The hypoglycemic activity of both plant extracts was compared with standard oral hypoglycemic treatment glibenclamide. These plants are considered as a perfect candidate for further investigations for justifying the modes of hypoglycemic effect, as well as for isolation and determination of phytochemicals that possess or are responsible for the hypoglycemic and anti-diabetic activity of the plant. Further extensive pharmaceutical surveys, including long term studies, could be of value to estimate the possible toxic activity of these plants.\n\n\nData availability\n\nZenodo: Hypoglycemic Activity of Two Herbal Extracts, https://doi.org/10.5281/zenodo.370136223\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nSamad A, Shams MS, Ullah Z, et al.: Status of herbal medicines in the treatment of diabetes: a review. Curr Diabetes Rev. 2009; 5(2): 102–11. PubMed Abstract | Publisher Full Text\n\nDetournay B, Simon D, Guillausseau PJ, et al.: Chronic kidney disease in type 2 diabetes patients in France: prevalence, influence of glycaemic control and implications for the pharmacological management of diabetes. Diabetes Metab. 2012; 38(2): 102–12. PubMed Abstract | Publisher Full Text\n\nKumar P, Kale RK, Baquer NZ: Antihyperglycemic and protective effects of Trigonella foenum graecum seed powder on biochemical alterations in alloxan diabetic rats. Eur Rev Med Pharmacol Sci. 2012; 16 Suppl 3: 18–27. PubMed Abstract\n\nBaquer NZ, Kumar P, Taha A, et al.: Metabolic and molecular action of Trigonella foenum-graecum (fenugreek) and trace metals in experimental diabetic tissues. J Biosci. 2011; 36(2): 383–96. PubMed Abstract | Publisher Full Text\n\nAl-Khateeb E, Hamadi SA, Al-Hakeemi AAN, et al.: Hypoglycemic effect of trigonelline isolated from Iraqi fenugreek seeds in normal and alloxan-diabetic rabbits. Eur Sci J. 2012; 8(30): 16–24. Reference Source\n\nSankar P, Subhashree S, Sudharani S: Effect of Trigonella foenum-graecum seed powder on the antioxidant levels of high fat diet and low dose streptozotocin induced type II diabetic rats. Eur Rev Med Pharmacol Sci. 2012; 16 Suppl 3: 10–7. PubMed Abstract\n\nHamza N, Berke B, Cheze C, et al.: Preventive and curative effect of Trigonella foenum-graecum L. seeds in C57BL/6J models of type 2 diabetes induced by high-fat diet. J Ethnopharmacol. 2012; 142(2): 516–22. PubMed Abstract | Publisher Full Text\n\nBaloch AH, Rehman H, Ibrahim Z, et al.: The Biology of Balochistani Weed: Cyperus rotundus Linnaeus. A Review. Pure Appl Biol. 2015; 4(2): 171–80. Publisher Full Text\n\nPirzada AM, Ali HH, Naeem M, et al.: Cyperus rotundus L.: Traditional uses, phytochemistry, and pharmacological activities. J Ethnopharmacol. 2015; 174: 540–60. PubMed Abstract | Publisher Full Text\n\nKhamees AH, Abdulhussein AJ, Sahib HB, et al.: Anti-angiogenic and Antioxidant Activity of Iraqi Cyperus rotundus Ethanol Extract. Int J Pharmacol. 2018; 14(4): 546–52. Publisher Full Text\n\nMead R: The design of experiments: statistical principles for practical applications. Mead R, editor. New York, USA: Cambridge University Press; 1988. Reference Source\n\nArifin WN, Zahiruddin WM: Sample Size Calculation in Animal Studies Using Resource Equation Approach. Malays J Med Sci. 2017; 24(5): 101–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShah NA, Khan MR: Antidiabetic Effect of Sida cordata in Alloxan Induced Diabetic Rats. Biomed Res Int. 2014; 2014: 671294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzkudelski T: The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res. 2001; 50(6): 537–46. PubMed Abstract\n\nCandasamy M, Murthy TE, Gubiyappa KS, et al.: Alteration of glucose lowering effect of glibenclamide on single and multiple treatments with fenofibrate in experimental rats and rabbit models. J Basic Clin Pharm. 2014; 5(3): 62–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuhad AMS, Ekbal AK, Ali AS: Phytochemical investigation of Suaeda baccata (Chenopodiaceae). Al-Mustansiriyah Journal for Pharmaceutical Sciences. 2005; 2(2): 51–7. Reference Source\n\nSarker SD, Nahar L: An introduction to natural products isolation. Methods Mol Biol. 2012; 864: 1–25. PubMed Abstract | Publisher Full Text\n\nKhamees AH, Kadhim EJ, Sahib HB, et al.: In vitro Analysis of Antioxidant and Antimicrobial Activity of Iraqi Bryonia dioica. Int J Pharm Sci Rev Res. 2017; 43(1): 248–52. Reference Source\n\nElsner M, Guldbakke B, Tiedge M, et al.: Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin. Diabetologia. 2000; 43(12): 1528–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurcelin R, Eddouks M, Maury J, et al.: Excessive glucose production, rather than insulin resistance, accounts for hyperglycaemia in recent-onset streptozotocin-diabetic rats. Diabetologia. 1995; 38(3): 283–90. PubMed Abstract | Publisher Full Text\n\nPatra JC, Chua BH: Artificial neural network-based drug design for diabetes mellitus using flavonoids. J Comput Chem. 2011; 32(4): 555–67. PubMed Abstract | Publisher Full Text\n\nPrasath G, Subramanian S: Fisetin, A bioflavonoid ameliorates hyperglycemia in STZ-induced experimental diabetes in rats. Int J Pharm Sci Rev Res. 2011; 61: 68–74. Reference Source\n\nFawzi H: Hypoglycemic Activity of Two Herbal Extracts (Version Microsoft Excel Worksheet) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3701362"
}
|
[
{
"id": "66714",
"date": "17 Jul 2020",
"name": "Talasila Eswara Gopala Krishna Murthy",
"expertise": [
"Reviewer Expertise Pharmaceutical product development and Isolation of active principles from plant materials."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article may be considered for publication after major revision.\nTable 2 has been reproduced with permission from 'Khamees AH, 2017. In vitro Analysis of Antioxidant and Antimicrobial Activity of Iraqi Bryonia dioica.' The original references for these tests shall be specified.\n\nThe basis for selection of alcoholic extract shall be specified.\n\nThe criterion for dose selection shall be specified.\n\nThe percent reduction in blood glucose levels shall be specified.\n\nThe statistical interpretation of the data is required for better understanding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": []
},
{
"id": "71414",
"date": "28 Sep 2020",
"name": "Supriti Sarkar",
"expertise": [
"Reviewer Expertise Endocrinology. Reproductive endocrinology",
"Effects of Phytochemicals on Diabetes."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature? Answer: Partly. The work done here though was not complicated rather was over-simplified but presented clearly. However the review of literature was insufficient and not put accurately. That is explained as follows: 1, In the first Para, where general description of diabetic complications are stated, the reference no 1 is not suitable. There are many works to refer to for such generalised diabetic complications e.g. Lin and Sun (20101). Again, the hypoglycaemic activity of Fenugreek seeds has been studied by a number of works, few of such didn’t mention here, which need to be cited e.g. El-Wakf et al. (20152). One of the main and important components of fenugreek seed is 4-hydroxyisoleucine that was not mentioned here (Singh et al., 20103). The hypoglycaemic effects of Cyperus rotundus was not reviewed here, though other functional properties were mentioned. The authors can consult at least these references:1) Singh et al. (20154); 2) Al-Snafi (20165) and 3) The Medical Journal Of Tikrit University (2003);9(2).\n\nIs the study design appropriate and is the work technically sound? Answer: Partly. The study design followed here was appropriate but could add some more parameters to strengthen the hypoglycaemic effects of fenugreek seed as well as tubers of Cyperus rotundus. Many previous workers already experimentally evidenced the hypoglycaemic effects of both fenugreek seed and Cyperus rotundus. In respect of that the current study didn’t show any novel findings. The blood glucose could be estimated by any standard biochemical methods instead of instant glucometer.\n\nAre sufficient details of methods and analysis provided to allow replication by others? Answer: Partly. The way of administration of both fenugreek seed extract as well as Cyperus rotundus should need to be mentioned, whether these were gavaged or mixed with food? Dose of alloxan seems to be little high. Was a single dose of alloxan sufficient to maintain diabetic blood sugar till the end of the experiment up to the 6th week? Why is the dose of alloxan as described in Experimental outcome and Study design different? A little description of phytochemical analysis need to be described for each specimen. Did the extraction of phytochemicals from fenugreek seed follow the same reference method? If possible names of different phytochemicals as extracted from both plants can be put here, because types of alkaloids etc. present in these plants are already available.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Answer: Yes.\n\nAre all the source data underlying the results available to ensure full reproducibility? Answer: Partly. Few more parameters could be estimated if possible.\n\nAre the conclusions drawn adequately supported by the results? Answer: Partly. It would be better if the authors could do some other parameters like estimation of insulin from the serum samples of all studied groups. At least any enzymes of glucose metabolic pathway from liver like hexokinase could be measured to improve the findings more scientifically. The obtained results then could support the hypoglycaemic effects of both plants more strongly. As most of the results described here are already known.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-247
|
https://f1000research.com/articles/9-16/v1
|
13 Jan 20
|
{
"type": "Correspondence",
"title": "A role for MIR828 in pineapple fruit development",
"authors": [
"Christopher D. Rock"
],
"abstract": "Chen et al. (Nature Genet. 51: 1549–1558; Oct. 2019) sequenced Ananas comosus var. bracteatus accession CB5, cultivated for its bright pink-to-red colored fruit, and yellow-fleshed A. comosus accession F153, reporting an improved F153 reference assembly while annotating MICRORNA (MIRNA) loci and gene family expressions relevant to lignin and anthocyanin biosynthesis. An independent article (Xiong et al. Sci. Rep. 8: 1947; 2018) reported var. bracteatus MIRNAs but not MIR828, a negative regulator of anthocyanin and polyphenolics biosynthesis by targeting MYB transcription factors associated with UV light- and sugar-signaling in dicots. MIR828 has been reported in gymnosperms, Amborella (sister to flowering plants), and basal monocot orders Liliales, Asparagales, Zingiberales, Arecales, but not in the Poales, a sister order comprising grasses and ~3,000 species of bromeliads including pineapple. Here I show MIR828 exists in pineapple and directs post-transcriptional gene silencing of mRNAs encoding MYB family members with inferred function to regulate the conspicuous red fruit trait in var. bracteatus. MIR828 plesiomorphy (an ancient basal trait) may shed light on monocot apomorphic fruit development, postulated for 21 monocot families with fleshy fruits as due to homoplasy/convergence driven by tropical climate and/or enticements to vertebrate endozoic seed dispersers.",
"keywords": [
"anthocyanins",
"microRNAs",
"fruit development",
"RNA interference",
"evolution"
],
"content": "\n\nChen et al.1 sequenced Ananas comosus var. bracteatus accession CB5, cultivated for its bright pink-to-red colored fruit, and yellow-fleshed A. comosus accession F153, reporting an improved F153 reference assembly2 while annotating MICRORNA (MIRNA) loci2–4 and gene family expressions relevant to lignin and anthocyanin biosynthesis. An independent article reported var. bracteatus MIRNAs5 but not MIR8286, a negative regulator of anthocyanin and polyphenolics biosynthesis by targeting MYB transcription factors associated with UV light- and sugar-signaling in dicots7–8. MIR828 has been reported in gymnosperms8, Amborella (sister to flowering plants)9, and basal monocot orders Liliales8,10, Asparagales11, Zingiberales12,13, Arecales14, but not in the Poales, a sister order comprising grasses and ~3,000 species of bromeliads including pineapple15. Here I show MIR828 exists in pineapple and directs post-transcriptional gene silencing of mRNAs encoding MYB family members with inferred function to regulate the conspicuous red fruit trait in var. bracteatus. MIR828 plesiomorphy (an ancient basal trait) may shed light on monocot apomorphic fruit development, postulated for 21 monocot families with fleshy fruits as due to homoplasy/convergence driven by tropical climate and/or enticements to vertebrate endozoic seed dispersers16.\n\nThe astronomer Carl Sagan popularized the quip (a corollary of Occam’s Razor) “absence of evidence is NOT evidence of absence.” Taking a conservative approach applies especially to quantitative transcriptomics. For example, the existence of regulatory and post-transcriptional processes increase the complexity of non-coding RNA space, where annotation is sparse. Analysis of Chen et al’s A. comosus MD-2 cultivar leaf small RNA (sRNA) libraries and stranded RNA-seq libraries4 from flowers and fruits of F153 and CB5 genotypes establish the existence of aco-miR828 (Extended data: Figure S117) and pri-MIR828 expressions, with the novel observation that pri-MIR828 is properly transcribed in leaves, flowers and fruits yet ~50% of mature miR828 species abundance (0.3 reads per million in leaves; Extended data: Table S117) are 21 nt, while 93% of the equally abundant 22 nt species appear to have undergone non-templated 3’ uridylation of the 21 nt species18 (Extended data: Table S117). Analysis of independent bracteatus cultivar leaf sRNA libraries (NCBI SRA SRR5677552-7; 113.2 million reads)5 of unknown provenance relative to the subject CB5 genotype failed to identify any MIR828 reads.\n\nFortuitously, a unique aspect of miR828 is that despite its very low abundance, it has very high activity7,19 that serves as diagnostic. miR828 guides ARGONAUTE slicing of target MYB mRNAs within the deeply conserved SANT domain region8 by Watson-Crick complementarity, with consequent knock-on production of easily quantified DICER-mediated sense- and antisense 21 nt phased small-interfering RNAs (phasiRNAs) mapping downstream (3’) on target MYB transcripts. The improved F153 reference assembly1 contains two candidate miR828-targeted MYBs: Aco017254.1 (LG4), with two introns of 1113 and 1340 nts, and Aco020986.1 (LG14) without RNA-seq evidence of intron splicing, whereas the CB5 bracteatus genome only contains one Aco017254.1 homologous gene with RNA-seq splicing evidence for conserved introns of 1109 and 1332 nt (Extended data: Table S217). When phasiRNA expressions from leaf sRNA libraries4,5 are respectively mapped to the F153 and CB5 candidate MYB target mRNAs, it is apparent that F153 MYB transcripts clearly undergo miR828-guided slicing, evidenced by D1(+) phased siRNA reads mapping to the 10th nucleotide position of miR828 homology to the mRNA target, and unique sense and antisense secondary phasi-RNAs mapping precisely in multiples of 21 nt downstream from the detected slice sites (Figure 1; Extended data: Figure S2, Table S317). The CB5 reference genome target MYB locus Aco017254.1 homolog (contig tig00012294, CABWKS010000088.1:25590-28868rc) encodes two missense codons at residues 41 (M➔R) and 200 (N➔K compared to F153), four silent codon substitutions, and RNA-seq analysis reveals mis-annotation of the CB5 genome which lacks two Gs (at contig residues 28780 and 27566) that exist and result in a CB5 open reading frame of the same size as Aco017254.1 in F153, including six instead of seven trinucleotide GGC glycine codon repeats templated in the CB5 genome at residue 204 (Extended data: Table S217).\n\nConcatenated sRNA libraries were used as pseudo-degradome on grounds both sliced mRNAs and amplified diced dsRNAs are manifest in sRNA libraries7. Slicing is at nt10 from 5' end of miR828 (arrow, inset; same target sequence for Aco020986.1 at mRNA nt 115). Y axis is numbers of degradome reads in sRNA libraries mapped to cDNAs; red dot is the documented miR828 sliced sRNA species that sets the register for 3’ phasiRNA production.\n\nIn contrast to demonstrated post-transcriptional silencing activity of miR828 on target MYB expressions in F153 leaf samples (Figure 1), analysis of six bracteatus leaf sRNA libraries from an independent study5 did not provide any evidence of target MYB Aco017254.1 mRNA slicing or target phasiRNA accumulations (Extended data: Table S317). Taken together, subject to the caveat that the provenance of the bracteatus cultivar used for the sRNA analysis5 may be different than subject CB5 genotype, the data suggest there may be differences in expression and/or regulation of pri-MIR828, and/or target MYB Aco017254.1 between F153 yellow-fleshed versus CB5 red-fleshed genotypes. Figure 2 shows this is indeed the case in various tissues examined, with the evidence supporting higher pri-MIR828 expression in F153 ovules concordant with lower target MYB Aco017254.1 mRNA levels (Figure 2A), significant decreases over time from stage 1 early fruit development for target MYBs Aco017254.1 and Aco020986.1 in yellow-fleshed MD-2 cultivar (Figure 2B), whereas in contrast there is lower expression of pri-MIR828 concordant with sustained higher expression of Aco017254.1 target MYB than seen in MD-2 during the ripening stage of red-fleshed CB5 genotype (compare Figure 2C with 2B).\n\nA) Inverse relationship of pri-MIR828 (high) to target MYB Aco017254.1 (low) anthocyanin effector expressions in F153 ovules. Error bars are s.e.m., n=3 biological replicates. B) Down regulation of miR828-targeted MYB anthocyanin effectors during MD2 yellow-fleshed fruit development (n=1 per stage). Asterisk (*) denotes stage 1 significantly different than stages 2-8, p = 0.02 (Student’s two-sided t-test, equal variance assumed). Stage 1 error bars show the 95% confidence interval for significance. C) Apparent maintenance of positive anthocyanin effector MYB Aco017254.1 expression at “ripe” two-month stage of CB5 red-fleshed fruit development, concordant with trend of lower pri-MIR828 expression. Error bars are s.e.m., n=5 biological replicates.\n\nPlant development gives rise to an astounding complexity of shapes, colors, and functions that Darwin called ‘an abominable mystery’ in his efforts to integrate species complexity with the unifying theory of evolution. The observations reported here potentially offer insight into the conservation of a developmental control mechanism whereby miR828 in pineapple, like in dicots silences MYBs inferred to act as positive effectors of anthocyanin biosynthesis that could give rise to the red-fleshed trait in the bracteatus variety. Consistent with this view is the finding from genome-wide functional phylogenomics approaches that ARGONAUTE1, RNA-DEPENDENT RNA POLYMERASE6, and mRNA export factor homolog SILENCING DEFECTIVE5, each required for trans-acting siRNA accumulation, played significant roles in the evolution of monocot metabolic and developmental traits20,21. Also of interest in this context is the claim of bracteatus genome authors22 that convergent expansion in several Crassulacean Acid Metabolism (CAM) bromeliad lineages of XAP5 CIRCADIAN TIMEKEEPER/XCT, a nuclear-localized regulator of blue light responses23 and sRNA production24, supports evolution of the myriad metabolic and physiological transitions from C3 to CAM photosynthesis by duplication/differentiation of a highly pleiotropic effector22.\n\n\nData availability\n\nRNA-seq data1, CB5 reference genome GCA_902506285.1, and F153 improved assembly genome GCA_902162155.1 were downloaded from NCBI via BioProject accession code PRJEB33121. sRNA raw data5 for A. comosus var. bracteatus leaf libraries were downloaded from NCBI BioProject accession code PRJNA389361. Processed data4 for MD-2 leaf sRNA libraries Pn_Gr2am_1, Pn_Gr3pm_3, Pn_Gr4pm_1, Pn_Gr4pm_3, Pn_Gr6am_2, Pn_GrMid_3, Pn_Wh10am_2, Pn_Wh10pm_1, Pn_Wh1pm_1, Pn_Wh2am_1, Pn_Wh2am_2, Pn_Wh3pm_1, Pn_Wh3pm_2, Pn_Wh4am_1, Pn_Wh4pm_1, Pn_Wh6am_1, Pn_Wh6am_2, Pn_Wh6pm_1, Pn_Wh6pm_2, Pn_Wh8am_2, Pn_Wh8pm_2, Pn_WhMid_1, and Pn_WhMid_2 were downloaded from https://mpss.danforthcenter.org/dbs/index.php?SITE=pineapple_sRNA.\n\nThe original A. comosus F153 reference genome and cDNA fasta file ‘Acomosus_321_v3.cds.fa’ can be browsed online and downloaded at https://phytozome.jgi.doe.gov/25.\n\nFigshare: A role for MIR828 in pineapple fruit development, https://doi.org/10.6084/m9.figshare.11388051.v117\n\nThis project contains the following extended data:\n\nFigure S1. ShortStack graphical linear output of Aco-MIR828 hairpin structure and sRNA abundance evidence from 23 MD-2 leaf sRNA libraries.\n\nFigure S2. PhaseTank alignment output of leaf MD-2 sRNA libraries mapped to candidate miR828 target MYB cDNAs, F153 improved reference genome.\n\nTable S1: Evidences for pri-MIR828 expression in F153 and CB5 varieties.\n\nTable S2: RNA-seq evidence for miR828 target MYB expressions.\n\nTable S3: bowtie mapping of sRNAs to miR828 target MYB mRNAs\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nFastx-toolkit version 0.0.14 for trimming Illumina universal adapterAGATCGGAAGAGCACACGTCTGAACTCCAGTCA (fastx_clipper option -l 18; discards short reads) and fasta file manipulations (fastx_uncollapser to expand the pre-processed data4; fastq_to_fasta for inputs to CleaveLand) is available at http://hannonlab.cshl.edu/fastx_toolkit/. FastQC version 0.11.5 for quality control of fastq raw sequence data is available at https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. ShortStack26 version 3.8.5 (options --mincov 15 --foldsize 340; Extended data: Figure S1, Table S117) for comprehensive annotation and quantitation of MIRNAs and sRNA cluster phasing is available at https://github.com/MikeAxtell/ShortStack. CleaveLand27 version 4.4 for analysis of sRNA libraries as pseudo-degradome inputs to find sliced miRNA targets is available at https://github.com/MikeAxtell/CleaveLand4. PhaseTank28 version 1.0 for quantifying and aligning phasiRNAs to miRNA target mRNAs (Extended data: Figure S217) is available at http://phasetank.sourceforge.net/; the linux command sed -i ‘s/-/_x/’ was used to reconfigure fastx_collapser output from concatenated sRNA fasta library files to PhaseTank input style “>t1_xN” where N is number of collapsed reads). Magic-BLAST29 version 1.5.0 for RNA-seq fastq read alignment (Extended data: Table S2 row 4 for options parameters17) to reference genomes is available at https://ncbi.github.io/magicblast/. Blastn30 version 2.6.0 is available at ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/. Bowtie31 version 1.1.2 for short read alignment (option -v 1 to allow one mismatch) is available at http://bowtie-bio.sourceforge.net/manual.shtml. RNAfold32 web server was used for generating miR828 hairpin graphics (Extended data: Figure S117) at http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi.",
"appendix": "Acknowledgements\n\nThe author thanks the TTU High Performance Computer Center for support in use of the Quanah supercluster, and Sunitha Sukumaran for help with perl scripting.\n\n\nReferences\n\nChen LY, VanBuren R, Paris M, et al.: The bracteatus pineapple genome and domestication of clonally propagated crops. Nat Genet. 2019; 51(10): 1549–1558. PubMed Abstract | Publisher Full Text\n\nMing R, VanBuren R, Wai CM, et al.: The pineapple genome and the evolution of CAM photosynthesis. Nat Genet. 2015; 47(12): 1435–1442. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng Y, Li T, Xu Z, et al.: Identification of microRNAs, phasiRNAs and their targets in pineapple. Trop Plant Biol. 2016; 9: 176–186. Publisher Full Text\n\nWai CM, VanBuren R, Zhang J, et al.: Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple. Plant J. 2017; 92(1): 19–30. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoy S, Tripathi AM, Yadav A, et al.: Identification and expression analyses of miRNAs from two contrasting flower color cultivars of Canna by deep sequencing. PLoS One. 2016; 11(1): e0147499. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXin C, Liu W, Lin Q, et al.: Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development. Genomics. 2015; 105(4): 242–251. PubMed Abstract | Publisher Full Text\n\nBarrett CF, Baker WJ, Comer JR, et al.: Plastid genomes reveal support for deep phylogenetic relationships and extensive rate variation among palms and other commelinid monocots. New Phytol. 2016; 209(2): 855–870. PubMed Abstract | Publisher Full Text\n\nGivnish TJ, Pires JC, Graham SW, et al.: Repeated evolution of net venation and fleshy fruits among monocots in shaded habitats confirms a priori predictions: evidence from an ndhF phylogeny. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nCibrián-Jaramillo A, De la Torre-Bárcena JE, Lee EK, et al.: Using phylogenomic patterns and gene ontology to identify proteins of importance in plant evolution. Genome Biol Evol. 2010; 2: 225–239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe La Harpe M, Paris M, Hess J, et al.: Genomic footprints of repeated evolution of CAM photosynthesis in tillandsioid bromeliads. BioRxiv. preprint. 2018. Publisher Full Text\n\nMartin-Tryon EL, Harmer SL: XAP5 CIRCADIAN TIMEKEEPER coordinates light signals for proper timing of photomorphogenesis and the circadian clock in Arabidopsis. Plant Cell. 2008; 20(5): 1244–1259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFang X, Shi Y, Lu X, et al.: CMA33/XCT regulates small RNA production through modulating the transcription of Dicer-Like genes in arabidopsis. Mol Plant. 2015; 8(8): 1227–1236. PubMed Abstract | Publisher Full Text\n\nGoodstein DM, Shu S, Howson R, et al.: Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2011; 40(Database issue): D1178–D1186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson NR, Yeoh JM, Coruh C, et al.: Improved placement of multi-mapping small RNAs. G3 (Bethesda). 2016; 6(7): 2103–2111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAddo-Quaye C, Miller W, Axtell MJ: CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics. 2009; 25(1): 130–131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo Q, Qu X, Jin W: PhaseTank: genome-wide computational identification of phasiRNAs and their regulatory cascades. Bioinformatics. 2014; 31(2): 284–286. PubMed Abstract | Publisher Full Text\n\nBoratyn GM, Thierry-Mieg J, Thierry-Mieg D, et al.: Magic-BLAST, an accurate RNA-seq aligner for long and short reads. 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}
|
[
{
"id": "58563",
"date": "19 Feb 2020",
"name": "Brian D. Gregory",
"expertise": [
"Reviewer Expertise Plant RNA biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article presents new findings that support the existence of MIR828 in the pineapple genome and that this microRNA may be involved in regulating MYB target RNAs for silencing to affect fruit coloration differences between various pineapple species. The study is well described and provides some intriguing new insights that other plant scientists are likely to be interested in reading. The conclusions are well supported and these preliminary findings definitely hint that the hypothesis suggested by Dr. Rock that this microRNA – target interaction may affect fruit color in pineapple is definitely plausible. I only have minor suggestions for changes to the article which I list below:\nThe phrase “MYB expressions” should be changed to “MYB abundance” throughout the manuscript.\n\nThe phrase “phasiRNA accumulations” should be changed to “phasiRNA accumulation”.\n\nThe phrase “phylogenomics approaches” should be changed to “phylogenomic approaches”.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "5345",
"date": "27 Mar 2020",
"name": "Christopher Rock",
"role": "Author Response",
"response": "Suggested changes made in revised article"
}
]
},
{
"id": "60755",
"date": "24 Mar 2020",
"name": "Nathan Johnson",
"expertise": [
"Reviewer Expertise Small RNA analysis and annotation in plants."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article focuses on identifying the presence of a miRNA loci (MIR828) within several cultivars of pineapple. The author leverages prior sequencing data from two studies to annotate a very lowly expressed locus which is apparently MIR828, marking the first example of this miRNA family in Poales. Despite the low expression, the author supports the presence of the miRNA through identification of targeted mRNAs, shown through detection of secondary siRNAs resulting from cleavage by a 22-nt miRNA. They also provide some evidence to speculate about the role miR828 may play in pineapple coloration relative to development.\nMy background is in small RNA annotation and interpretation of function in plants. I believe that this article does a good job identifying a miRNA that is challenging to find in pineapple. I also think that the author does a compelling job proving its actual function through secondary siRNAs. False negatives are always present in the computational annotation of genomes and this type of careful analysis is often needed to find exceptions that are not caught by programs using default settings.\nI believe the conclusions of this article are true and the results to be adequate, pending some revision. My criticisms of this article mainly have to do with clarity. I think that several changes must be made to make the article more transparent in the analyses performed, the language used to describe it, and the presentation of data. I do not suggest any additional experiments to support their result, but I would like a response and modification to the manuscript based on the following notes:\n“Analysis of Chen et al’s A. comosus MD-2 cultivar leaf small RNA (sRNA) libraries and stranded RNA-seq libraries from flowers and fruits of F153 and CB5 genotypes establish the existence of aco-miR828 (Extended data: Figure S1) and pri-MIR828 expressions, with the novel observation that pri-MIR828 is properly transcribed in leaves, flowers and fruits yet ~50% of mature miR828 species abundance (0.3 reads per million in leaves; Extended data: Table S1) are 21 nt, while 93% of the equally abundant 22 nt species appear to have undergone non-templated 3’ uridylation of the 21 nt species (Extended data: Table S1).” --- This sentence does not clearly describe what “analysis” has taken place. I need to see the author describe what evidence supports the 3 conclusions listed here, both in writing and with a description of the analysis pipeline in the methods section (see below).\n\nIt is preferred to include a genome-browser shot showing the read coverage for the pri-miRNA regions listed in supp figure 1, as it can be difficult to identify from a table what the locus actually looks like for the reader. It also appears that many of the rna-seq reads originate from the ‘minus’ strand. This is surprising, as the hairpin is never double stranded. Could you explain this discrepancy?\n\nI like that the ShortStack output is included in supp. table 1. However, it is not clear to me what each locus refers to. What is the reference genome for each of these? It appears that cluster_1381 and cluster_1104 are using the Chen et al. sequencing data on the older reference genome. Why have these been shown and can you explain the lack of this locus in the older genome? I’m not sure what these loci are referring to compared to the first locus shown (cluster_4352).\n\nYour RPMs for this miRNA locus are extremely low (0.3 RPM), more than 10 fold lower than ShortStacks default locus identification threshold (5 RPM). This is a real lack and makes it challenging to identify this as a miRNA. Lacking a mir-star is also a problem, but might not be surprising for a low-expression locus. As this is a published miRNA you are identifying, I am inclined to agree that it is real. However, it is inappropriate to include an artificial sequence to make ShortStack call it as such, as is done in Supp. Figure 1. Please correct this and clearly identify that this locus lacks mir-star support, though is still likely a miRNA.\n\nConsider looking into the package strucVis, an add-on for RNAfold which shows nucleotide-resolution read depth on the folded diagram. I think this would greatly clarify this figure.\n\n“Analysis of independent bracteatus cultivar leaf sRNA libraries (NCBI SRA SRR5677552-7; 113.2 million reads) of unknown provenance relative to the subject CB5 genotype failed to identify any MIR828 reads.” --- This is surprising! Did you also try analysis using the F153 reference, considering the provenance of this variety is unknown? I think this is important considering the considerable depth of these libraries, where we might expect around 30 reads coming from this locus. Despite the lack of a locus identified, it might be useful to look directly at the alignment through a browser, to see if any small RNAs are coming from this locus.\n\n“...mature miR828 species abundance (0.3 reads per million in leaves; Extended data: Table S1)...” --- Please show the approximate read depth here also, as was shown with the Xiong et al libraries.\n\nFigure 1 is labeled as a degradome, when it is actually a plot of 5’ ends of sRNA alignments, thought to be secondary siRNAs. While I agree that secondary siRNAs can be strong evidence of targeting. However, this is entirely distinct from a degradome, which are derived from isolated mRNA degradation products. Please remove references of this as a degradome and correct the language to clearly describe this. This also should contain a citation for one of the following papers: where this evidence was used for confirming targeting (Shahid et al. 20181), or evidence of secondary sRNAs as a product of miRNA targeting (Allen et al. 20052).\n\nFigure 2 has several critical problems. In general, I think the author must provide more detail in the conclusions and evidence shown in each section of the figure. I have detailed some more specific points as follows:\nIt is deceiving to use bar plots to represent such small numbers of data points. Bars that represent an N of ideally 5 or less are much more informative shown as points representing the actual data. To show error bars (I assume... it is not indicated if these are standard error or deviation) greatly misrepresents the variance of these data. I would like to see these figures redone showing points rather than bars, or at least showing the raw data in a supplementary table (see below).\n\nFigure 2B is vague in its description of the test, and replicates involved. The legend describes an N=1 for all stages. If this is true, how can a statistical comparison be made? Stage 1 shows error bars, does this represent N>1? Additionally, precisely which samples are being grouped and compared here for the test? This must be made clearer.\n\nThe interpretation of results in Figure 2C is unclear. There is little to no textual description of what the author concludes from Figure 2 in general and it is mainly confined to one sentence where all three sub-figures are mentioned. This needs to be greatly expanded upon for me to understand what they perceive as the outcome of these analyses.\n\nI recommend including *all* of the raw data shown in these charts in supplementary tables in addition to a more clear demonstration of the data-points mentioned above. This allows the reader to clearly identify what is being shown in each.\n\nA description of methods is lacking. Though the sequencing data is derived from prior publications, the analysis performed in this paper is new. You need to describe the logical steps taken for each analysis, including processing, tools used in a step, the actual commands used, and data that is output. This would go a long way to clarifying the actual analyses they performed. This needs to be shown for RNA-seq analyses, sRNA-seq analyses, and identification of pri-miRNAs.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Partly\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "5346",
"date": "07 Apr 2020",
"name": "Christopher Rock",
"role": "Author Response",
"response": "Author responses to verbatim copied comments from Reviewer2 below, in conjunction with revised article submission.“Analysis of Chen et al’s A. comosus MD-2 cultivar leaf small RNA (sRNA) libraries and stranded RNA-seq libraries from flowers and fruits of F153 and CB5 genotypes establish the existence of aco-miR828 (Extended data: Figure S1) and pri-MIR828 expressions, with the novel observation that pri-MIR828 is properly transcribed in leaves, flowers and fruits yet ~50% of mature miR828 species abundance (0.3 reads per million in leaves; Extended data: Table S1) are 21 nt, while 93% of the equally abundant 22 nt species appear to have undergone non-templated 3’ uridylation of the 21 nt species (Extended data: Table S1).” --- This sentence does not clearly describe what “analysis” has taken place. I need to see the author describe what evidence supports the 3 conclusions listed here, both in writing and with a description of the analysis pipeline in the methods section (see below).RESPONSE: I thank the Reviewer for thorough and careful review and suggestions for improvement. Journal proscription on word limit (1,000) for the manuscript body (including Methods) was the reason for the (overly) brief description(s). Here is a pedantic description of the logical analyses employed to make the three referenced aco-MIR828 expression conclusions. Conclusion 1 “existence of aco-miR828 (Fig. S1)”: is from two independent ShortStack expression analyses detailed in Suppl. Table S1; i) mapping of sRNAs to the candidate aco-MIR828 locus/primary-MIR828 transcript corresponding to ShortStack Cluster_43525 in the new improved F153 reference genome, and ii) phasing of secondary siRNAs mapped as clusters to target MYB mRNA/cDNAs (older F153 annotated genome) triggered by inferred aco-miR828-programmed AGO slicing activity. Fig. S1 shows default ShortStack graphical aco-MIR828 hairpin secondary structure; the legend refers to Suppl. Table S1 for ShortStack tabular output details. Those tabular outputs on row 11 (primary-MIR828 Cluster_43525, length 333 nt) shows “Major RNA species”= UCUUGCUCAAAUGAGUAUUCCU, which is grounds for the claimed 3’ non-templated uridylated species (see elaboration below in Conclusion3 response). Tabular and graphical ShortStack outputs in Suppl. Table S1 and Fig. S1, respectively, are intended to document parameters required, according to community standards, for MIRNA annotation (Axtell MJ and Meyers BC [2018] “Revisiting criteria for plant microRNA annotation in the era of Big Data” Plant Cell 30: 272–284). Suppl. Table S1 rows 13 and 14 document ShortStack parameters of phased secondary siRNA clusters derived from two MYB target mRNAs/cDNAs: row13 “MajorRNA species” is D2(+) 21mer in phase (phasing score=20.2) with miR828 slice site activity independently substantiated by CleaveLand degradome analysis detailed in Fig. 1 (left panel). Furthermore, Fig. S2 further substantiates independent algorithmic evidence by PhaseTank software for the siRNA phasing on both MYB mRNA/cDNA targets triggered by miR828 activity. Suppl. Table S1 Row 14 “MajorRNA species”= CUAGGAGUGAAAGAUUACGCC was subsequently found (and annotated as such in a revised Suppl. Table S1) to map exactly 12 registers downstream from the 10th nt position of D2(-) phasiRNA shown in Fig. S2 for mRNA/cDNA Aco020986. Thus, this additional subsequent analysis supports that Aco020986 D2(-) phasiRNA may analogously program AGO for secondary slicing of Aco020986 mRNA/cDNA and help explain the low ShortStack phasing score of 2.9 for Aco020986. Another plausible reason for the low phasing score of Aco020986 versus Aco017254 is the ~50% fewer reads (drawn from the multi-mapper functionality of ShortStack, parameter “RPM”) and higher reads complexity for Aco020986 per ShortStack output parameters. Notwithstanding, CleaveLand (Fig. 1 right panel) degradome and PhaseTank algorithmic analyses (Suppl. Fig. S2) independently show Aco020986 MYB mRNA/cDNA is a target of aco-miR828, substantiating not only aco-miR828 existence from expression data, but also its function.Conclusion 2 “existence [and proper transcription] of pri-MIR828 expressions [by analysis] of stranded RNA-seq libraries from flowers, and fruits of F153 and CB5 genotypes”: See Suppl. Table S1, rows 17-52 output parameters from MagicBLAST analysis. Row 22 is singular evidence of an RNA-seq read in genotype F153 leaf tissue (not formally claimed). Rows 23-36 are reads from F153 ovules and stamens. Rows 37- 52 are reads from genotype CB5 fruit.Conclusion 3 “~50% of mature miR828 species abundance are 21 nt, while 93% of the equally abundant 22 nt species appear to have undergone non-templated 3’ uridylation of the 21 nt species”: See above re: row 11 of Suppl. Table S1 that establishes the MajorRNA species expressed in the sRNA libraries is the hypothesized non-templated 22 nt aco-miR828. Note that ShortStack output reports five reads of 20 nt size, 29 reads of 21 nt size, and 35 reads of 22mer size. Thus at face value assuming all species of these sizes correspond to mature miR828s (see below), then 51% would be the miR828 22mer species (other ShortStack-reported 23 and 24 nt sizes are few and discounted, yet documented in Suppl. Table S1). Rows 54- 136 of Suppl. Table S1 detail every sRNA read in the subject libraries sorted by position having at most one mismatch aligned by bowtie to the F153 primary-MIR828 locus corresponding to ShortStack full Cluster_43525, including 113 nt upstream and 97 nt downstream of the miR828 hairpin shown in Suppl. Fig. S1 as default ShortStack output. Each relevant read is annotated manually as 20mer mature miR828, 21mer mature miR828, 22mer perfect match, or 22mer non-perfect matches to mature miR828. There are four 20mer mature miR828, 26 21mer mature miR828, one perfect match mature miR828 22mer, one 22mer with a “C” mismatch at nt 22, and 28 22mers with “T” mismatch at nt 22 of miR828 that is the claimed non-templated 3’ uridylated species. Thus (26 +4)/(26+4+30) = 50% 20mer plus 21mer mature miR828 (my claim of “~50% 21 mer”), and 93% of 22mer species are 3’ uridylated (28/30). It takes >700 words here to pedantically articulate the grounds that prove the compound claims in that one sentence of the manuscript text.It is preferred to include a genome-browser shot showing the read coverage for the pri-miRNA regions listed in supp figure 1, as it can be difficult to identify from a table what the locus actually looks like for the reader. It also appears that many of the rna-seq reads originate from the ‘minus’ strand. This is surprising, as the hairpin is never double stranded. Could you explain this discrepancy?RESPONSE: I have modified Suppl. Fig. S1, keeping the default ShortStack dataset graphical output (instead of the suggested browser screenshot) but adding rows for species to visualize all relevant data described above for bowtie output in Suppl. Table S1, rows 57-136, where parameters of strand and importantly sRNA read location on the primary MIR828 transcript are detailed, along with manual annotations (including for nine anti-sense sRNA reads mapping only up- and downstream of the hairpin per se). Note also that formally speaking, the Reviewer’s reference to the ‘pri-miRNA regions’ at issue is most analogous to the “pre-MIRNA” after ‘stem-to-loop’ biogenesis as described in Adams, L. [2017] “Pri-miRNA processing: structure is key.” Nat. Rev. Genet. 18: 145) which is the default output of ShortStack software showing only the minimal foldback encompassing expression reads for mature miR828 and miR828-star. Suppl. Fig. S1 was verbatim output from ShortStack software that automatically trims the foldback stem distal to the miRNA/miRNA-star duplex. In this matter the full length cluster #43525 corresponding to the ‘primary-MIRNA’ transcript is described in Suppl. Fig. S1 image header as “Cluster_43525 Original Location: CABGUK010000004.1:12043665-12043997 Displayed Location: CABGUK010000004.1:12043762-12043883 Strand: -“. Therefore the graphical presentation in Suppl. Fig. S1 represents those pertinent sRNA species detailed in the Suppl. Table S1 list of reads corresponding to pri-MIRNA transcript positions 114 to 235. The bowtie-mapped reads described in Suppl. Table S1, rows 57- 136 sorted by primary-MIR828 position are now fully accounted for in the revised Suppl. Fig. S1 which retains the graphical default ShortStack output style. I added to the original Suppl. Fig. S1 four additional rows for the other species that are germane to the issue (excluding only four 23mer reads that have non-templated “CU” residues at the 3’ terminus of miR828, where the second “U” residue could be templated thus confounding description). Now both graphical (revised Suppl. Fig. S1) and tabular representations (Suppl. Table S1) quantify the principal species at issue: the 20 and 21 nt mature miR828 (~50%), the 22 nt mature miR828 with either a non-templated “T” as the last residue (lower case; ~50% of all reads), a non-templated “C”, or a perfect match 22 nt template mature miR828, and a single phased read (in the terminal loop) upstream of the predicted miR828-star species (shown all lowercase).Regarding the RNA-seq antisense issue, note that row 20 of Suppl. Table S1 has a disclaimer description which the Reviewer may have overlooked: “Read 2 is the confirmed sense mRNA strand for CB5 datasets, based on an abundant transcript (bromelain RD21A, Aco011478.1) described by Chen et al. authors, used as control. The F153 ovule and stamen libraries are unstranded, based on the same control gene assessment.” It was deduced empirically from the RD21A reference gene characterization that the authors used un-stranded RNA-Seq kits early in the chronology of the project when many of the F153 ovule and stamen libraries were prepared and sequenced using different Illumina platforms. As evidence to support this interpretation, note there only exists a forward strand “read 1” for leaf, ovule, and stamen SRA datasets ERR3413090 and ERR3413005-8 (most are read lengths 1 x 100 bp, supporting their earlier chronology), despite being incorrectly annotated at NCBI SRA archive as “paired read” libraries. Specifically, rows 27, 28, and 36 are antisense strand reads challenged by the Reviewer and accounted for in the row 20 note as a computational artifact, thus counted as proper sense reads based on evidence from a control gene RD21A. For rows 38, 39, 46, 48, and 51, which are from CB5 fruit tissues (SRA datasets ERR3412993-3413002; read lengths 2 x 150 bp indicating newer Illumina chemistry and chronology) the libraries are verified empirically by expression evidence to be stranded paired read libraries. Note that in these antisense read cases, every read is from the antisense direction (read 1) of paired reads, as described in the row 20 disclaimer note.I like that the ShortStack output is included in supp. table 1. However, it is not clear to me what each locus refers to. What is the reference genome for each of these? It appears that cluster_1381 and cluster_1104 are using the Chen et al. sequencing data on the older reference genome. Why have these been shown and can you explain the lack of this locus in the older genome? I’m not sure what these loci are referring to compared to the first locus shown (cluster_4352).RESPONSE: as described above for “Conclusion1” evidence, row 11 aco-MIR828 ShortStack output is based on the new improved F153 reference genome made available as BioProject accession code PRJEB33121. ShortStack output for Rows 13 and 14 clusters were drawn from the cDNA fasta file of the original F153 reference genome available for download in phytozome12 as described in Data availability. Thus the two ShortStack analyses are different and cluster numbers are arbitrary. The reason why the older cDNA reference set from phytozome12 was used is because the new improved F153 reference genome does not have any cDNA annotation reference set. See https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/902/162/155/GCA_902162155.1_F153/.Your RPMs for this miRNA locus are extremely low (0.3 RPM), more than 10 fold lower than ShortStacks default locus identification threshold (5 RPM). This is a real lack and makes it challenging to identify this as a miRNA. Lacking a mir-star is also a problem, but might not be surprising for a low-expression locus. As this is a published miRNA you are identifying, I am inclined to agree that it is real. However, it is inappropriate to include an artificial sequence to make ShortStack call it as such, as is done in Supp. Figure 1. Please correct this and clearly identify that this locus lacks mir-star support, though is still likely a miRNA.RESPONSE: I have revised Suppl. Fig. S1 legend to state “This locus formally lacks miR828-star support.” Suppl. Fig. S1 legend clearly explained the miR828-star species was not sequenced from the dataset, but rather was seeded manually to drive ShortStack to formally call the MIRNA as “Yes” by default parameters, instead of “N15” in which case no graphical ShortStack output like Suppl. Fig. S1 is possible. It is argued that the thought experiment as described and presented is appropriate, if it can be justified. The Reviewer will recognize, as one skilled in the art of ShortStack, that without seeding a miR828-star sequence into the dataset, there would be no graphical ShortStack output at all, now revised (see above point) to meet the Reviewer’s specification to show the bona fide small RNA reads topology and abundances. The justification for the miR828-star “thought experiment” was to ensure annotation took into account default folding and trimming parameters built into the software where outputs are styled to objectively meet community standards for evidence-based MIRNA annotation. The reason for the exercise was not to obfuscate the truth that the star species was not found (which is clearly disclosed and the Reviewer concedes is not fatal because this is deeply conserved plant MIRNA), but rather to address a potential criticism that the “bulged” distal loop tripartite secondary structure (Suppl. Fig. S1) is an atypical feature of MIRNA foldbacks that could be an important determinant for Dicer recognition as substrate. This concern is grounded in the possibility a plant-specific “terminal loop-to-base” non-canonical processing sequence operates on aco-MIR828, like known for ath-miR156a biogenesis. Candidate aco-miR828 has a loop-side bulge analogous to the three nt loop-side bulge in ath-miR156a which appears as a “kink” in the foldback and could be argued as potentially critical for duplex biogenesis (Kim et al. [2016] “Structural determinants of miR156a precursor processing in temperature-responsive flowering in Arabidopsis.” J. Exp. Bot. 67: 4659). Being able to pass all algorithmic criteria as demonstrated by successful ShortStack annotation of candidate aco-MIR828 (conceding the miR828-star aspect) discounts theoretical concerns about a “bulged” distal loop tripartite secondary structure possibly antagonizing Dicer duplex biogenesis at the locus.Consider looking into the package strucVis, an add-on for RNAfold which shows nucleotide-resolution read depth on the folded diagram. I think this would greatly clarify this figure.RESPONSE: I have revised Suppl. Fig. S1 to add the suggested strucVis image of small RNA read depth coverage across the aco-primary-MIR828 locus encompassing the entire ShortStack reads Cluster_43525.“Analysis of independent bracteatus cultivar leaf sRNA libraries (NCBI SRA SRR5677552-7; 113.2 million reads) of unknown provenance relative to the subject CB5 genotype failed to identify any MIR828 reads.” --- This is surprising! Did you also try analysis using the F153 reference, considering the provenance of this variety is unknown? I think this is important considering the considerable depth of these libraries, where we might expect around 30 reads coming from this locus. Despite the lack of a locus identified, it might be useful to look directly at the alignment through a browser, to see if any small RNAs are coming from this locus.RESPONSE: Note that rows 18 and 19 of Suppl. Table S1 state: “subject database F153 pri-MIR828 genome coordinates: CABGUK010000004.1:12043665-12043997rc; subject database CB5 pri-MIR828 genome coordinates: CABWKS010000025.1:21332774-21333104rc.” I have compared these two sequences by BLASTing the entire F153 aco-MIR828 locus and they are identical except for a single nucleotide polymorphism on the 3’ leg of the foldback, far downstream of the miR828-star at position 195 of the alignment (mature miR828 and miR828-star underlined). Thus the F153 and CB5 loci are identical for the purposes of running ShortStack and mapping small RNAs from bracteatus genotype sources.Please see here. I have also subsequently run the strucVis software on the CB5 bam-indexed ShortStack output, as suggested to look directly at the alignment: there is no evidence of any reads mapping from the independent bracteatus cultivar leaf sRNA libraries to the CB5 MIR828, as claimed (data not shown). This is despite ShortStack calling 52 bona fide MIRNAs (47 21mers, five 22mers), compared with 29 MIRNAs (24 21mers, five 22mers) called with the F153 small RNA dataset.“...mature miR828 species abundance (0.3 reads per million in leaves; Extended data: Table S1)...” --- Please show the approximate read depth here also, as was shown with the Xiong et al libraries.RESPONSE: The answer is 264.7 million reads, available in Suppl. Table S1, row 5; that information has been added accordingly to the text.Figure 1 is labeled as a degradome, when it is actually a plot of 5’ ends of sRNA alignments, thought to be secondary siRNAs. While I agree that secondary siRNAs can be strong evidence of targeting. However, this is entirely distinct from a degradome, which are derived from isolated mRNA degradation products. Please remove references of this as a degradome and correct the language to clearly describe this. This also should contain a citation for one of the following papers: where this evidence was used for confirming targeting (Shahid et al. 20181), or evidence of secondary sRNAs as a product of miRNA targeting (Allen et al. 20052). References1. Shahid S, Kim G, Johnson N, Wafula E, et al.: MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 2018; 553 (7686): 82-85 Publisher Full Text2. Allen E, Xie Z, Gustafson AM, Carrington JC: microRNA-directed phasing during trans-acting siRNA biogenesis in plants.Cell. 2005; 121 (2): 207-21 PubMed Abstract | Publisher Full TextRESPONSE: I have revised the text as advised, calling inputs ‘pseudo-degradome’ amplified sense and antisense siRNAs derived from miRNA-sliced mRNAs, and citing those two priority references in addition to the 2019 self citation #7 where we coined the term pseudo-degradome in a study of vvi-miR828 activity.Figure 2 has several critical problems. In general, I think the author must provide more detail in the conclusions and evidence shown in each section of the figure. I have detailed some more specific points as follows:It is deceiving to use bar plots to represent such small numbers of data points. Bars that represent an N of ideally 5 or less are much more informative shown as points representing the actual data. To show error bars (I assume... it is not indicated if these are standard error or deviation) greatly misrepresents the variance of these data. I would like to see these figures redone showing points rather than bars, or at least showing the raw data in a supplementary table (see below).RESPONSE: A factor perhaps not considered by the Reviewer when asking for “whisker plots,” i.e. drawn from separate data points: note that library size/depth determine normalized data points per library sample, summed within each library and reported as the mean of three biological replicate libraries. Thus only three such data points could possibly be shown for such a requested whisker plot, despite the underlying basis being many independent data points making up the normalized reads per library. Error bars were expressly described as standard error of three biological replicates in the legend of Fig. 2. The legend has been revised to direct readers to the raw data which was provided in Suppl. Table S1 rows 23-36 for MIR828 (panel A: 11 independent data points for ovules, 3 data points for stamens), and Suppl. Table S2, rows 406-470 for MYB mRNAs (panel A: 11 independent data points for Aco017254 ovules, 28 for stamens; 3 data points for Aco020986 ovules, 11 for stamens). It is rebutted that bars are thus not representing N < 5 data points in Fig. 2A as critiqued except for two: MIR828 stamens and Aco020986 ovules. It is conceded the error bars are not compelling statistically, but pointed out that no claims are made for significance while maintaining data integrity by reporting observed biological variance as found in the data. The verbatim claim made from Fig. 2A data states: “evidence supports higher pri-MIR828 expression in F153 ovules concordant with lower target MYB Aco017254.1 mRNA levels.” This qualified claim is grounded from 11 independent ovule MIR828 and Aco017254 data points, respectively, averaged across three biological replicates which is derivative of the experimental design as found and interpreted conservatively. Figure 2B is vague in its description of the test, and replicates involved. The legend describes an N=1 for all stages. If this is true, how can a statistical comparison be made? Stage 1 shows error bars, does this represent N>1? Additionally, precisely which samples are being grouped and compared here for the test? This must be made clearer.RESPONSE: The Reviewer is appreciated for due diligence and critical insights. Here are the raw independent reads data summarized from Suppl. Table S2, rows 134-405, which the reader is directed to in the revised legend for completeness: Aco017254 stages 1, 2, 3, 4, 5, 7 and 8 have respectively 38, 37, 45,11, 5, 6, and 26 data points; Aco020986 has respectively 6, 7, 7, 0, 0, 1, and 3 unique independent data points (not cross- mapped to homologous Aco017254) for stages 1-8. The statistical comparison is made by taking independent abundances for Aco017254 and Aco020986 in stage 1 MD2 fruit as one condition matrix, and stages 2-8 as the second paired condition matrix across both genes to conduct a paired two-sided Student’s t-test for significance; the result is p = 0.02. The legend has been revised to clarify as requested. Stage 1 “error” bars in question are actually calculated 95% confidence intervals for hypothetical significance if there were a second biological replicate (extant experimental dataset is n=1) by using an unpaired Student’s t-test and introducing a second matrix datapoint for each gene of a magnitude sufficient to give p = 0.05. That magnitude, determined empirically, is the boundary marked by the low error bar as the 95% percentile confidence interval for statistical significance. The purpose of the analysis is to graphically show by an independent estimation, consistent with the paired t-test actual result, that stages 2-8 means are all well below the lower error bar confidence interval for significance calculated for stage 1.The interpretation of results in Figure 2C is unclear. There is little to no textual description of what the author concludes from Figure 2 in general and it is mainly confined to one sentence where all three sub-figures are mentioned. This needs to be greatly expanded upon for me to understand what they perceive as the outcome of these analyses. I recommend including *all* of the raw data shown in these charts in supplementary tables in addition to a more clear demonstration of the data-points mentioned above. This allows the reader to clearly identify what is being shown in each.RESPONSE: Again it is noted the issue of terse description of interpretations was driven by publisher proscription on word limits. Here are the raw independent reads data summarized from Suppl. Table S1 rows 37-52 for MIR828 and Suppl. Table S2, rows 471-883 for CB5 Aco017254, referenced in a revised Fig. 2 legend: 8 MIR828 independent data points each for 1 month- and 2 month-old CB5 fruits, and 171 data points for 1 month old-, and 222 data points for 2 month-old Aco017254 expression in CB5 fruits. Here is restated a revised but nonetheless succinct paraphrased claim from the text that intends to clarify how to compare evidences in Fig. 2C with Fig. 2B: “there may be differences in expression and/or regulation of pri-MIR828, and/or target MYB Aco017254.1 between F153 yellow-fleshed versus CB5 red-fleshed genotypes…. there is a trend of lower expression of CB5 pri-MIR828 concordant with sustained higher abundance of Aco017254.1 target MYB (Figure 2C stage 7 versus stage 1) than seen in MD-2 during the ripening stage of red-fleshed CB5 genotype (compare Figure 2C stage 7 showing high CB5 Aco017254.1 abundance to Figure 2B stage 7 showing very low MD-2 Aco017254.1 abundance).”A description of methods is lacking. Though the sequencing data is derived from prior publications, the analysis performed in this paper is new. You need to describe the logical steps taken for each analysis, including processing, tools used in a step, the actual commands used, and data that is output. This would go a long way to clarifying the actual analyses they performed. This needs to be shown for RNA-seq analyses, sRNA-seq analyses, and identification of pri-miRNAs.RESPONSE: This issue was raised by the journal’s managing Editor at the submission stage and addressed by the author. A statement is added to the end of the Software availability and methods section “The options parameters used for various algorithms are detailed in Extended data: Tables S1- S3.”"
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https://f1000research.com/articles/9-16
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https://f1000research.com/articles/9-245/v1
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08 Apr 20
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{
"type": "Case Report",
"title": "Case Report: Acute hydrotherapy with super-saturated hydrogen-rich water for ankle sprain in a professional athlete",
"authors": [
"Dejan Javorac",
"Valdemar Stajer",
"Sergej Ostojic",
"Dejan Javorac",
"Valdemar Stajer"
],
"abstract": "Background: The traditional treatment of soft tissue injuries consists of the RICE protocol – rest, ice, compression, and elevation, followed for up to 72 hours after a trauma. Although designed as an immediate therapy to reduce inflammation that occurs after an acute injury, the RICE protcol might not be the best way to promote healing due to limiting blood flow. Molecular hydrogen (H2) has recently been put forward as a possible adjuvant treatment in musculoskeletal medicine, yet limited data are available concerning its effectiveness as a first-aid intervention. Case report: We report here a case of an elite professional athlete who suffered a grade II ankle sprain, and who subsequently received six sessions of ankle and foot hydrotherapy (e.g. 30-min at every four hours) with super-saturated hydrogen-rich water during the first 24 hours post-injury. The pain VAS self‐completed by the patient dropped from 50 points (moderate pain) at baseline (immediately after injury) to 20 points (mild pain) at 24-h follow-up. Ankle swelling dropped by 2.8% and dorsiflexion range of movement improved by 27.9% from baseline to follow-up, respectively. Conclusions: Our case has indicated that an acute multi-session hydrotherapy with hydrogen-rich water might be a helpful treatment in terms of pain, swelling reduction and regaining range of motion after an ankle sprain.",
"keywords": [
"molecular hydrogen",
"ankle sprain",
"hydrotherapy"
],
"content": "Introduction\n\nSoft tissue injuries (STIs) remain among the most prevalent traumata in musculoskeletal medicine1. Sprains, strains and contusions are common STIs that often occur during sport and exercise activities2. STIs usually require immediate treatment to reduce inflammation, bleeding and damage within the injured tissue (e.g. muscle, tendon, ligaments, joint), with management options typically comprise a break from the activity that caused an injury, and different physical therapy procedures. Applying rest, ice, compression, and elevation (RICE) therapy is referred as a long standard first-aid treatment protocol for STIs3. However, insufficient evidence appears to be available to determine the relative effectiveness of RICE therapy for specific STIs4. In particular, RICE therapy might not be the best way to promote tissue healing due to limiting blood flow, and alternative methods and techniques are advocated to help manage STIs5. Among others, molecular hydrogen (H2) has recently been put forward as a possible adjuvant treatment in musculoskeletal medicine6. Addition of oral and topical H2 intervention to RICE protocol was effective to reduce inflammation and augment range-of-motion recovery in athletes who suffered a soft tissue injury7. However, no data are available concerning the effectiveness of hydrogen when applied as an individual first-aid intervention. This case report illustrates the efficacy and safety of acute multi-session hydrotherapy with super-saturated hydrogen-rich water for ankle sprain in a professional male athlete.\n\n\nCase report\n\nWe are reporting here the case of a 29 year old male Caucasian professional football athlete who suffered a sport-related ankle sprain in April 2019. The injury occurred during a regular exercise session on artificial turf as an inversion sprain accompanied by plantar flexion. The patient was immediately evaluated by a sports medicine specialist who confirmed the category and the degree of injury (grade II ankle sprain) by physical examination. The patient was an apparently healthy young men (age 29 years, weight 77.0 kg, height 184.0 cm, professional experience 11 years), with no history of ankle sprain (or other lower extremity injuries) in the past 6 months, and no cardiometabolic or other musculoskeletal disorders. Written informed consent was obtained from the patient in accordance with the Declaration of Helsinki, and study protocol approved by the local IRB at the FSPE Applied Bioenergetics Lab at the University of Novi Sad (A14-2019).\n\nAt the initial examination immediately after the injury, right ankle was painful, swollen and warm, and had increased laxity on testing (Figure 1, Panel a). Self-completed visual analog scale (VAS) score for pain was 50 points (moderate pain). Figure-of-eight method of measuring ankle joint swelling at the injured ankle revealed 56.5 cm, with weight-bearing lunge test (WBLT) showing 43.1 mm. The patient demonstrated a diminished ability to bear weight.\n\nAn image of sprained ankle: (a) Immediately after the injury; (b) At 24-h follow-up after six 30-min sessions of hydrotherapy with super-saturated hydrogen-rich water.\n\nHydrotherapy with super-saturated hydrogen-rich water was used as an exclusive treatment (besides rest) with the main aim to reduce symptoms and signs of acute ankle sprain, and it was hoped to speed up the acute recovery. Super-saturated hydrogen-rich water was produced by putting a magnesium-producing formulation (10 g) into a 3-L stationary whirlpool with tap water of neutral temperature (20°C). Hydrogen was produced by a following reaction: Mg + H2O → H2 + Mg(OH)2, with concentration of hydrogen in a whirlpool ~ 8 ppm. The intervention was provided by HRW Natural Health Products Inc. (catalogue number 6-27843; New Westminster, BC, Canada). Throughout the 24 hours after the ankle sprain, the participant received six 30-min ankle baths (e.g. one hydrotherapy every 4 hours), with the first session given immediately after an initial examination (~ 60 min after the injury). During each session, the foot and ankle of injured leg were immersed in a stationary whirlpool. All hydrotherapies were formulated and supervised by a health care professional.\n\nAt the 24-h follow-up examination (Figure 1, Panel b), VAS score for pain dropped to 20 points (mild pain), with ankle circumference decreased to 54.9 cm; WBLT improved to 55.0 mm. The patient reported no side effects of hydrogen intervention (e.g. pain, cramps, tingling, discoloration of skin, burning, itching, rash), as evaluated with open-ended questionnaire administered at the end of each session of hydrotherapy, and at 24-h follow-up.\n\n\nDiscussion\n\nThis case report suggests the beneficial effects of hydrotherapy with hydrogen-rich water as a possible treatment to decrease pain and swelling in a professional athlete with moderate-degree ankle sprain. A number of recent animal and human studies evaluated the efficacy of H2 in musculoskeletal medicine. H2 appears to be effective in tackling disuse muscle atrophy in rats8, Duchenne muscular dystrophy in mice9, bone loss induced by modeled microgravity10 or ovariectomy-induced osteoporosis in rats11, and mitigate disease activity in patients with rheumatoid arthritis12. For acute injuries, hydrogen-rich saline (1 ml/100 g) seems to be beneficial in attenuating muscle damage in a rat model of skeletal muscle injury induced by 3-h tourniquet occlusion and 4-h reperfusion13. Our group reported advantageous effects of 2-week administration of topical and oral H2 (added to the RICE therapy) on plasma viscosity and functional recovery in a randomized controlled trial with professional athletes who suffered STIs7. In this case report, we found that acute hydrotherapy with super-saturated hydrogen-rich water might be beneficial when used as the exclussive first-aid treatment in specific STIs, such as ankle sprain. H2 positively affected STI-induced signs and symptoms at 24-h follow up perhaps due to its antioxidant, anti-inflammatory and anti-apoptotic effects14. Due to its small size and higher-grade diffusibility6, topical hydrogen could be easily transported to hard-to-reach tissues, including areas of injury that are often characterized by low drug penetrability15. Specifically, H2 might prevent (or offset) the generation of toxic compounds that occur after initial injury due to subsequent cell damage and tissue hypoxia, often referred as a secondary injury16.\n\nThis professional athlete received a rather intensive treatment, with H2 hydrotherapy applied in several recurrent episodes throughout the first 24 hours post-injury, a pattern that might be highly applicable to an athlete looking for an accelerated and efficacious strategy in STIs management and recovery3. This treatment dynamics appear to be comparable to the traditional acute RICE protocol for STIs medical care in terms of frequency (e.g. number of sessions per day) and duration of intervention (e.g. length of individual session)4. On the other hand, acute H2 hydrotherapy may be superior to RICE protocol due to improved blood flow (as implied here by a decreased swelling at follow-up), while the ice component of RICE reduces blood flow to the injured area and delays healing5. To confirm this hypothesis, future case series should compare two interventions in a double-blind, parallel-group randomized controlled design, by evaluating peripheral circulation at the site of injury.\n\nDue to the fact that hydrogen in water tends to evaporate over time17, hydrogen-rich water for every session was prepared fresh and administered for 30 min. A report have shown that gaseous hydrogen remains detained in water exposed to air for up to 2 hours before its concentration drops below a therapeutical level (e.g. < 1 ppm)18. The super-saturated hydrogen-rich water used here (8 ppm) perhaps provides an advantage of supraphysiological dosages of H2 administered in a time-optimized manner. However, how H2 concentration changes during the session of hydrotherapy remains unknown. Despite that, topical H2 hydrotherapy yielded no side effects in our patient, confirming an affirmative safety record for H2 reported by others [for review see Ref. 16].\n\nSeveral limitations must be considered when the study findings are interpreted. First, we report a relatively short period of the intervention and post-injury assessment (e.g. 24 hours) while no medium- and long-term efficacy and safety of topical hydrogen were evaluated. Second, only limited compendium of clinician- and patient-reported outcomes were analyzed, while no biomarkers of tissue injury or inflammation were employed, and a possible mechanism of hydrogen action remains unknown. Finally, it remains open to question how topical H2 positively affects the acute recovery of other STIs with different location, etiology and severity.\n\n\nConclusions\n\nEven though our results are based on a single-patient report, our case has indicated that an acute multi-session hydrotherapy with hydrogen-rich water might be a safe and helpful treatment in terms of pain, swelling reduction, and regaining the range of motion after an ankle sprain. We suggest a closer monitoring of the efficacy and safety of topical H2 therapy use in different musculoskeletal injuries on a larger similar case series.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report, including any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "References\n\nCollins M, September AV, Posthumus M: Biological variation in musculoskeletal injuries: current knowledge, future research and practical implications. Br J Sports Med. 2015; 49(23): 1497–1503. PubMed Abstract | Publisher Full Text\n\nPatel DR, Yamasaki A, Brown K: Epidemiology of sports-related musculoskeletal injuries in young athletes in United States. Transl Pediatr. 2017; 6(3): 160–166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolfe MW, Uhl TL, Mattacola CG, et al.: Management of ankle sprains. Am Fam Physician. 2001; 63(1): 93–104. PubMed Abstract\n\nvan den Bekerom MP, Struijs PA, Blankevoort L, et al.: What is the evidence for rest, ice, compression, and elevation therapy in the treatment of ankle sprains in adults? J Athl Train. 2012; 47(4): 435–443. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBleakley C, McDonough S, MacAuley D: The use of ice in the treatment of acute soft-tissue injury: a systematic review of randomized controlled trials. Am J Sports Med. 2004; 32(1): 251–261. PubMed Abstract | Publisher Full Text\n\nOstojic SM: Should hydrogen therapy be included in a musculoskeletal medicine routine? [version 1; peer review: 2 approved]. F1000Res. 2016; 5: 2659. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOstojic SM, Vukomanovic B, Calleja-Gonzalez J, et al.: Effectiveness of oral and topical hydrogen for sports-related soft tissue injuries. Postgrad Med. 2014; 126(5): 187–195. PubMed Abstract | Publisher Full Text\n\nFujita R, Tanaka Y, Saihara Y, et al.: Effect of molecular hydrogen saturated alkaline electrolyzed water on disuse muscle atrophy in gastrocnemius muscle. J Physiol Anthropol. 2011; 30(5): 195–201. PubMed Abstract | Publisher Full Text\n\nHasegawa S, Ito M, Fukami M, et al.: Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice. Redox Rep. 2017; 22(1): 26–34. PubMed Abstract | Publisher Full Text\n\nSun Y, Shuang F, Chen DM, et al.: Treatment of hydrogen molecule abates oxidative stress and alleviates bone loss induced by modeled microgravity in rats. Osteoporos Int. 2013; 24(3): 969–978. PubMed Abstract | Publisher Full Text\n\nGuo JD, Li L, Shi YM, et al.: Hydrogen water consumption prevents osteopenia in ovariectomized rat. Br J Pharmacol. 2013; 168(6): 1412–1420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIshibashi T, Sato B, Rikitake M, et al.: Consumption of water containing a high concentration of molecular hydrogen reduces oxidative stress and disease activity in patients with rheumatoid arthritis: an open-label pilot study. Med Gas Res. 2012; 2(1): 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang T, Wang W, Tu C, et al.: Hydrogen-rich Saline Attenuates Ischemia-Reperfusion Injury in Skeletal Muscle. J Surg Res. 2015; 194(2): 471–480. PubMed Abstract | Publisher Full Text\n\nOhta S: Recent progress toward hydrogen medicine: potential of molecular hydrogen for preventive and therapeutic applications. Curr Pharm Des. 2011; 17(22): 2241–252. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuckwalter J, Woo SL: Drug treatment of soft tissue injuries efficacy and tissue effects. Iowa Orthop J. 1993; 13: 40–48. PubMed Abstract | Free Full Text\n\nOstojic SM: Molecular hydrogen in sports medicine: new therapeutic perspectives. Int J Sports Med. 2015; 36(4): 273–279. PubMed Abstract | Publisher Full Text\n\nHuang CS, Kawamura T, Toyoda Y, et al.: Recent advances in hydrogen research as a therapeutic medical gas. Free Radic Res. 2010; 44(9): 971–982. PubMed Abstract | Publisher Full Text\n\nMolecular Hydrogen Institute: Concentration and solubility of H2. Half life of H2 in a solution. (Assessed on 18 April 2019). Reference Source"
}
|
[
{
"id": "62119",
"date": "21 Apr 2020",
"name": "Steven R. Brenner",
"expertise": [
"Reviewer Expertise 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\nI thought there was good documentation of the injury including physical examination, photographs and pain scales.\n\nThe application of hydrogen water as treatment with reference to schedule and treatment application was well documented and should be repeatable.\n\nThe ankle injury was to a professional athlete. Was the athlete able to return to athletic competition as soon utilizing the hydrogen water method as would be possible using the more traditional “RICE”, approach to treatment of ankle injuries, primarily sprained ankle injury?\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": "5471",
"date": "30 Apr 2020",
"name": "Sergej Ostojic",
"role": "Author Response",
"response": "Thank you for your comment. We haven't been able to evaluate post-injury recovery in terms of return to athletic competition due to the nature of this study, However, this issue requires an additional trial since this aspect might be of great importance for professional athletes who usually strive for re-getting to competition as soon as possible."
}
]
},
{
"id": "62120",
"date": "30 Apr 2020",
"name": "Cheong Hwa Ooi",
"expertise": [
"Reviewer Expertise Exercise Physiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this case report argue that there is insufficient evidence on the effectiveness of RICE therapy for specific soft tissue injuries. Hence, further development of the intervention for soft tissue injuries is warranted. With that, they presented a case of a professional footballer who suffered a grade II ankle sprain and subsequently treated with six 30-minute foot immersion in hydrogen-rich water (3 L, ~8 ppm). The author concluded that the treatment might be safe and helpful in reducing pain and swelling, and regaining the range of motion after an ankle sprain.\n\nIt seems that it is the left ankle, as shown in Figure 1, but the authors reported it as right ankle sprain in the text. Further clarification is needed. The treatment was given every 4 hours throughout the 24 hours after injury, and it is reasonable to assume that the nighttime sleep would have been disrupted. Does it represent a feasible and ideal therapy for the athlete? The water temperature for the foot/ankle bath was 20°C, how does adding hydrogen-producing magnesium tablets into it help the blood flow? Further explanation of the mechanism for improved blood flow after the treatment is relevant to the understanding of hydrogen medicine in soft tissue injury.\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": "5470",
"date": "30 Apr 2020",
"name": "Sergej Ostojic",
"role": "Author Response",
"response": "Thank you for your valuable comments and notes. We confirmed that the right ankle was injured yet we depicted here a mirror image. Also, the intensive hydrogen treatment might disrupt sleeping time but that's the case with all intensive measures for acute ankle injuries since the treatment requires immediate and intensive care during the firts 24 to 48 h do to specific pathophysiology. Further studies are highly warranted to evaluate the possible effects of hydrogen on blood flow, a factor that might contribute to its mechanism(s) of action."
}
]
}
] | 1
|
https://f1000research.com/articles/9-245
|
https://f1000research.com/articles/9-244/v1
|
07 Apr 20
|
{
"type": "Research Article",
"title": "Knowledge and Self-use of Medicinal Plants by Health University Students in Brasília-Brazil",
"authors": [
"Anna Maly de Leão e Neves Eduardo",
"Guilherme Junio Pinheiro",
"Erica Carine Campos Caldas Rosa",
"Eleuza Rodrigues Machado",
"Alexis Fonseca Welker",
"Guilherme Junio Pinheiro",
"Erica Carine Campos Caldas Rosa",
"Eleuza Rodrigues Machado",
"Alexis Fonseca Welker"
],
"abstract": "Background: This article reports an experiment based on the measurement of the academic about the use of medicinal plants is increasing, this article aims to analyze the behavior of future health professionals regarding the use of medicinal plants, since they will be responsible for the orientation and education of patients and the population. Methods: The present study involved 50 students of the academic health care Anhanguera College of Brasilia (Biological Sciences, Biomedicine, Nursing and Pharmacy), selected randomly. Questions about the use of medicinal plants were made and 96% confirmed having used medicinal plants. We used the Descriptive statistic to compare the answers. Results: Most academics reported self-consumption of medicinal plants, with a positive result after use (*p<0.001). The most used medicinal plants were, in addition to the mixture of herbs, boldo and Mexican tea, for the treatment of stomach pains, headaches, coughs, and colds. Self-usage was mainly due to the recommendation of a family member and most of their knowledge of herbal medicine come from non-evidence based sources. The prevalence of plant use was higher among females(84%) in all undergraduate courses observed when purchased as males (22%). Conclusions: The consumption of medicinal plants among academics is high, but it is within the literature. However, as the study was conducted among health professionals, they were expected to have greater knowledge of the adverse effects and on the plant to be used.",
"keywords": [
"Medicinal plant",
"Academic health",
"Consumption",
"Knowledge",
"Students",
"Herbal"
],
"content": "Introduction\n\nThe consumption of plants for medicinal purposes has increased in recent years, a tradition that has been passed from generation to generation. Medicinal plants have therapeutic properties, causing various effects on the human body, induced by phytochemicals. However, several professionals do not learn phytotherapy/herbal medicine as undergraduate students (Jamshed et al., 2016; Lapidus, 2007; Oliveira et al., 2012; Suchard et al., 2004). Different studies showed that physicians and university students studying health related courses do not have adequate knowledge of medicinal plants (Ameade et al., 2015; Frass et al., 2012; Hina et al., 2015; Jamshed et al., 2016; Johnson et al., 2008; Oliveira et al., 2012; Samara et al., 2019; Suchard et al., 2004). Despite this, several studies showed a high level of self-medication with herbal medicines by health students and professionals (Abahussain et al., 2007; Ambrose & Samuels, 2004; Awad et al., 2012; Brambila-Tapia et al., 2016; El Ezz & Ez-Elarab, 2011; Fahmy et al., 2010; Gyasi et al., 2016; Saha et al., 2017; Sawalha et al., 2008; Soroush et al., 2018). This practice is worrying because some of the biologically active compounds of plant origin can be harmful, and cause some adverse and toxic effects (Cheung et al., 2009; Krenzelok & Mrvos, 2011; Ng et al., 2019; Pan et al., 2017; Sriapha et al., 2015; Zamawe et al., 2018). To date, it is not clearly known whether these students search for scientific evidence about herbs before consuming them. Therefore, we seek to analyze the knowledge of university students in health related courses on medicinal plants and their self-use.\n\n\nMethods\n\nTo analyze the knowledge and self-use of medicinal plants, a cross-sectional study was performed with university students in health-related courses. The participants were recruited and selected as follows. Students that were at the university and outside of the classrooms were informed about the intention to conduct the study. The sample size was determined by the number of undergraduate students that: (i) agreed to participate in the study and signed the written informed consent, and met the inclusion criteria. The inclusion criteria for the research participants were being students regularly enrolled in the educational institution, being between 19 and 50 years old, agreeing to participate in the research, signing the Free and Informed Consent Form, and being present on the day of the interview. As a criterion for exclusion from the research were academics who did not agree to participate in the research or to sign the Informed Consent Form.\n\nThe students who participated in the research were studying higher education at a private educational institution, in Brasília (Brazil), in the following health courses: Biomedicine (13), Biological Sciences (07), Nursing (13) and Pharmacy (17), coming to 50 students total.\n\nA questionnaire was developed based on earlier studies (Ambrose & Samuels, 2004; Ameade et al., 2015; Nworu et al., 2015) specifically for this study to assess students' knowledge and self-use of medicinal plants. After students signed the Informed Consent form, the questionnaires were administered from September 2014 to February 2015. The data collection instrument consisted of objective questions and was structured in two sections: in the first, the profile of the interviewees (age, course, and sex) is described; in the second, information related to the use of medicinal plants was described, such as the plants used, the result obtained after consumption, adverse events, previous knowledge about plants, research for knowledge about the plants before their use, and the motivation for the use of medicinal plants. The questionnaire was applied through an interview that lasted between 10 to 15 minutes. Consent forms and the study questionnaire are provided as extended data (Eduardo et al., 2020).\n\nThis research project was approved by the Research Ethics Committee, registered by the Research Ethics Committees (CEPs) and by the National Research Ethics Committee (CONEP), also known as the CEP / CONEP system (approval number 34629314.2.0000.5372). Written informed consent from all subjects involved was obtained for participation in the study.\n\nStatistical analysis\n\nThe IBM Statistical Package for the Social Sciences (IBM 21) and the GraphPad 8.1 Prisma software were used to analyze data (Fisher’s tests) on the knowledge and use of medicinal plants by health students (Brambila-Tapia et al., 2016; Eduardo et al., 2019). For all analyses, p values below 0.01 were considered statistically significant.\n\n\nResults\n\nDescriptive data\n\nThe sample size of this study was 50 university students in the health field, 84% of which were female and 16% male. Gender distributions across courses are displayed in Figure 1. The semesters that students were taking in undergraduate health courses were: Biomedicine course - 14% were in the 7th semester and 12% in the 8th semester; Biological Sciences - 2% were in the 3rd semester and 12% were in the 4th; Nursing course - 2% were in the 1st semester and 24% were in the 5th semester; and Pharmacy course - 34% were in the 8th semester, as shown in Table 1 (see underlying data (Eduardo et al., 2020)).\n\nOf the total of 50 students, 25 of them were aged 19 to 29, 13 were aged 30 to 39, 18 aged 40 to 49, and 4 aged 50 to 59 (Figure 2). Results show that most respondents 48 (96%; P <0.0001), had at some point in their life used medicinal plants for the alternative treatment of diseases. Of these, 46 (P <0.0001) reported they cured their illnesses, and only 4% did not report a positive effect after using medicinal plants (Figure 3A). In addition, of the individuals who used medicinal plants, 54% said that before using them, they researched the plants, for information such as the indications for use and the adverse effects that they can trigger, whereas 42% do not usually research the plants before ingesting them (Figure 3B). Most of the interviewees (68%) reported that used medicinal plant based on the recommendation of family members (Figure 4).\n\nDistribution of students who had and did not have positive effects on the use of medicinal plants (Figure 3A). Figure 3B shows the distribution of academics who usually research the medicinal plant before ingesting it in the years 2014 and 2015.\n\nRegarding the motivations that led to the consumption of medicinal plants, most reported stomach pain (30%), followed by headache, cough, colds, and pain in general (Figure 5).\n\nThe medicinal plants most used by academics are shown in Figure 6, where 44% of them mentioned they use a mixture of plants, such as chamomile (Matricaria chamomilla), lemon balm (Melissa officinalis), fennel (Pimpinella anisum) and pomegranate (Punica granatum), 30% said they use the boldo (Peumus boldus), 12% mentioned the Mexican tea (Dysphania ambrosioides). Raw survey results are available as Underlying data (Eduardo et al., 2020).\n\n\nDiscussion\n\nThis study showed that although a high percentage of undergraduate students from a private health institution were able to know medicinal plants and use them, a significant fraction did not research before consumption. Only a minority did not obtain a positive result after consuming medicinal plants. These data corroborate the results obtained by several authors, which indicated the effectiveness of using medicinal plants for several problems such as constipation, colds, cough and bacterial infections and pain in general (Ameade et al., 2015; Lee et al., 2005; Nworu et al., 2015; Soroush et al., 2018).\n\nRegarding the indication of plants consumed by students, most of them reported following family traditions. Other studies also showed the influence of family members or friends on the consumption of natural products (Ambrose & Samuels, 2004; Ameade et al., 2015; Gyasi et al., 2016; Lee et al., 2005; Nworu et al., 2015; Saha et al., 2017), particularly parents and grandparents (Oliveira et al., 2012; Otieno & Analo, 2012). These non-academic sources of information and the lack of courses on herbal medicine at the college (Jamshed et al., 2016; Lapidus, 2007; Oliveira et al., 2012; Suchard et al., 2004) may explain why physicians and other health professionals present inadequate knowledge of medicinal plants (Ameade et al., 2015; Frass et al., 2012; Hina et al., 2015; Jamshed et al., 2016; Johnson et al., 2008; Oliveira et al., 2012; Samara et al., 2019; Suchard et al., 2004). When herbal medicine is not taught, the belief that herbal treatment is an effective method becomes lower in senior medical students in relation to younger/1st year students (Akan et al., 2012; Beglaryan & Amirjanyan, 2012; Brambila-Tapia et al., 2016). Consequently, several studies showed a poor self-perceived competence or confidence of physicians and pharmacists in dealing with herbal medicines (Abahussain et al., 2007; Erku et al., 2017; Kemper et al., 2006; Xu & Levine, 2008). Some studies showed that undergraduate health students who take herbal courses – either because it is mandatory or a well-attended elective – show an increase in the level of knowledge about natural products, herb properties and usage (Cai & Boyd, 2018; Johnson et al., 2008; Karpa, 2012; Larrañeta, 2018; McKennon et al., 2018). However, the level of interest for learning this subject may be low. Medical students have reported poor interest in complementary and alternative medicine (CAM) (Frass et al., 2012), whereas pharmacy students have shown a strong interest in traditional Chinese medicine and medicinal herbalism (Freyman et al., 2006).\n\nThe high level of students’ self-medication/usage in the present study is in accordance with other studies (Abahussain et al., 2007; Ambrose & Samuels, 2004; Awad et al., 2012; Brambila-Tapia et al., 2016; El Ezz & Ez-Elarab, 2011; Fahmy et al., 2010; Gyasi et al., 2016; Saha et al., 2017; Sawalha et al., 2008; Soroush et al., 2018). University students studying health related courses use at least one herbal substance per year (Johnson & Blanchard, 2006). Actually, this practice may be didactic/instructive since self-experimentation has been shown to improve student learning in relation to active learning approaches (Eduardo et al., 2019).\n\nOur finding that the self-administration of medicinal herbs occurs mostly without searching for more knowledge before their ingestion is in accordance with another study that showed a low percentage of university students that learned from personal research (Mackowiak et al., 2001). Health care professionals use more herbs for which there is scientific evidence of effectiveness (Gardiner et al., 2006) and health students believe one of the main barrier that limit the use of CAM is the lack/deficiency of scientific evidence (Awad et al., 2012; Brambila-Tapia et al., 2016; James & Bah, 2014; Saha et al., 2017; Samara et al., 2019; Zimmerman & Kandiah, 2012). Various articles investigating the source of knowledge/information about herbal medicine and related therapies did not mention scientific journals/magazines (Kilic et al., 2009). A recent study showed that pharmacy students gain must of their information about herbal medicine from non-scientific sources (Ashraf et al., 2019). Actually, scientific jounals and academic materials have been classified by university students as of low importance in terms of sources of knowledge regarding herbal medicine-related information (Gyasi et al., 2016) and less than one third of medical students use scientific magazines/journals as sources of information on medicinal plants (Samara et al., 2019). Other recent studies showed that medical and pharmacy students use social media and the internet as the main source of information about herbal medicine (Ashraf et al., 2019; James & Bah, 2014; Kilic et al., 2009; Nworu et al., 2015; Samara et al., 2019; Soroush et al., 2018). As a consequence, health professionals’ knowledge about medicinal plants do not come from scientific evidence (Frass et al., 2012; Oliveira et al., 2012). This non-scientific knowledge tend to be propagated by professionals, since consumers often consider pharmacists as a major source of information (Jamshed et al., 2016; Siti et al., 2009). An exception was observed in a study with chinese nursing students, which reported books, news papers and magazines as the most common sources of knowledge (Mei-Ying et al., 2004). The low interest of health students in seeking scientific information on medicinal plants is worrying because pharmacologically active compounds of natural origin may cause side effects, intoxication/poisoning and death (Cheung et al., 2009; Krenzelok & Mrvos, 2011; Ng et al., 2019; Pan et al., 2017; Sriapha et al., 2015; Zamawe et al., 2018). As discussed before, the offering of courses may improve the knowledge of students. However, courses offered at the university may use non-scientific information. The inclusion of evidence-based information about herbs/CAM into the curriculum in some medical and nursing schools failed to meet accepted standards of evidence-based medicine; the curricula on the integrative medicine were strongly biased in favor of CAM and many references were poor-quality clinical trials (Marcus & McCullough, 2009). A study reported that even after teaching CAM using evidence-based medicine, students showed only a modest increase on their skill level in understanding the medical literature (Forjuoh et al., 2003). However, courses of herbal/natural product that include bibliographic instruction on information seeking/searching skills increase students’ knowledge about reputable resources for evidence-based information on medicinal herbs (Diaz-Cruz & Bolten, 2016; Dvorkin et al., 2004; Karpa, 2012; Lapidus, 2007; McKennon et al., 2018).Students that are more familiar with PubMed/Medline databases reported that herbal medicine is applicable to their work (Owen & Fang, 2003). The participation in courses that teach how to search for evidence-based resources regarding herbal medicines make students gain confidence in responding to patient questions (McKennon et al., 2018). Additionally, courses underlying the use of natural products that are improved over the years – providing evidence-based medicine – have been shown to increase health students’ skills in providing advice to patients (Geldenhuys et al., 2015). These literature data together with our results corroborate the theory that evidence-based courses of herbal medicine should be offered more to students in health related courses at universities.\n\nThe responses of the students regarding the symptoms of the diseases treated with medicinal plants, there was a predominance of stomach pain, headache, and cough, however, some mentioned fever and pain in general. For these ailments, they used a mix of herbs such as chamomile, lemongrass, fennel, and pomegranate, or individual plants such as boldo and Mexican tea. These results corroborate with those obtained from a School of Pharmacy in London, where students mentioned the use of individual herbal remedies and mixed herbal remedies such as ginger, saffron, chamomile and arnica (Freymann et al., 2006), with some plants also mentioned in our study.\n\nOur results indicate that a high proportion of health university students do not research before self-usage of medicinal plants and that most of their knowledge come from non-evidence based sources. This study had some limitations, the main one being the number of students who wanted to participate in the research. Although our sample size was larger than those of other studies (Soroush et al., 2018; Xu & Levine, 2008), the findings of the present study may not reflect general attitudes, and do not permit the generalizability of our results to all university students in health related courses, even specifically to Brazil. Another limitation of our study is that only health students were investigated. Due to the high prevalence of self-medication among different students of other courses, we consider it important that new studies assess and compare the perceived factors of self-medication among students of different areas.\n\nWe conclude that the self-consumption of medicinal plants by academics in health related courses of a private institution in Brasilia is high, with the practice stimulated mainly by family members. However, knowledge of adverse reactions may be limited, as a high proportion of the students do not research the plants before using them. Moreover, most of their knowledge of medicine herbs comes from non-evidence based sources. The use of an evidence-based teaching tool that addresses medicinal plants could be positive for university students in health sciences, as being able to provide information to the population is an essential skill.\n\n\nData availability\n\nFigshare: Eduardo et al., 2020 Dataset_Knowledge and self-use of medicinal plants by health university students in Brasília-Brazil. https://doi.org/10.6084/m9.figshare.11591913.v5 (Eduardo et al., 2020)\n\nThis project contains the following underlying data:\n\nEduardo et al., 2020 dataset1.xls.xlsx (students’ responses to the questionnaire)\n\nFigshare: Eduardo et al., 2020 Dataset_Knowledge and self-use of medicinal plants by health university students in Brasília-Brazil. https://doi.org/10.6084/m9.figshare.11591913.v5 (Eduardo et al., 2020)\n\nThis project contains the following extended data:\n\nEduardo et al., 2020 Questionnaire - plants.docx (questionnaire)\n\nEduardo et al., 2020 Informed Consent - plants.docx (written informed consent)\n\nEduardo et al., 2020 Approval by the research ethics committee - plants.docx (approval by the research ethics committee)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe thank the students who participated in the study, the Anhanguera College of Brasilia, and all those who collaborated in the analysis and conduct of the research.\n\n\nReferences\n\nAbahussain NA, Abahussain EA, Al-Oumi FM: Pharmacists' attitudes and awareness towards the use and safety of herbs in Kuwait. Pharm Pract (Granada). 2007; 5(3): 125–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkan H, Izbirak G, Kaspar EC, et al.: Knowledge and attitudes towards complementary and alternative medicine among medical students in Turkey. BMC Complement Altern Med. 2012; 12: 115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmbrose ET, Samuels S: Perception and use of herbals among students and their practitioners in a university setting. J Am Acad Nurse Pract. 2004; 16(4): 166–73. PubMed Abstract | Publisher Full Text\n\nAmeade EP, Amalba A, Helegbe GK, et al.: Medical students' knowledge and attitude towards complementary and alternative medicine - A survey in Ghana. J Tradit Complement Med. 2015; 6(3): 230–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshraf M, Saeed H, Saleem Z, et al.: A cross-sectional assessment of knowledge, attitudes and self-perceived effectiveness of complementary and alternative medicine among pharmacy and non-pharmacy university students. BMC Complement Altern Med. 2019; 19(1): 95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAwad AI, Al-Ajmi S, Waheedi MA: Knowledge, perceptions and attitudes toward complementary and alternative therapies among Kuwaiti medical and pharmacy students. Med Princ Pract. 2012; 21(4): 350–4. PubMed Abstract | Publisher Full Text\n\nBeglaryan M, Amirjanyan A: Analysis of consumer demand for herbal medicine in the Republic of Armenia. Georgian Med News. 2012; (203): 61–7. PubMed Abstract\n\nBrambila-Tapia AJ, Rios-Gonzalez BE, Lopez-Barragan L, et al.: Attitudes, Knowledge, Use, and Recommendation of Complementary and Alternative Medicine by Health Professionals in Western Mexico. Explore (NY). 2016; 12(3): 180–7. PubMed Abstract | Publisher Full Text\n\nCai Y, Boyd DL: Effect of a traditional Chinese medicine course for undergraduate nursing students: A pre-/post-test study. Nurse Educ Today. 2018; 70: 87–93. PubMed Abstract | Publisher Full Text\n\nCheung WI, Tse ML, Ngan T, et al.: Liver injury associated with the use of Fructus Psoraleae (Bol-gol-zhee or Bu-gu-zhi) and its related proprietary medicine. Clin Toxicol (Phila). 2009; 47(7): 683–5. PubMed Abstract | Publisher Full Text\n\nDiaz-Cruz ES, Bolten BC: An elective course to enhance students’ knowledge and confidence in natural products. Currents Pharm Teach Learn. 2016; 8(5): 688–697. Publisher Full Text\n\nDvorkin L, Gardiner P, Whelan JS: Herbal medicine course within pharmacy curriculum. J Herb Pharmacother. 2004; 4(2): 47–58. PubMed Abstract | Publisher Full Text\n\nEduardo AMLN, Rosa ECCC, Welker AF: Effects of self-experimentation during practical classes on student learning [version 1; peer review: awaiting peer review]. F1000Res. 2019; 8: 1623. Publisher Full Text\n\nEduardo AMLN, Pinheiro G, Rosa ECCC, et al.: Eduardo et al 2020 Dataset_Knowledge and self-use of medicinal plants by health university students in Brasília-Brazil. figshare. Dataset. 2020. https://figshare.com/articles/Eduardo_et_al_2020_Dataset/11591913\n\nEl Ezz NF, Ez-Elarab HS: Knowledge, attitude and practice of medical students towards self medication at Ain Shams University, Egypt. J Prev Med Hyg. 2011; 52(4): 196–200. PubMed Abstract\n\nErku DA, Belachew SA, Mekuria AB, et al.: The role of community pharmacists in patient counseling and health education: a survey of their knowledge and level of involvement in relation to type 2 diabetes mellitus. Integr Pharm Res Pract. 2017; 6: 137–143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFahmy SA, Abdu S, Abuelkhair M: Pharmacists' attitude, perceptions and knowledge towards the use of herbal products in Abu Dhabi, United Arab Emirates. Pharm Pract (Granada). 2010; 8(2): 109–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForjuoh SN, Rascoe TG, Symm B, et al.: Teaching medical students complementary and alternative medicine using evidence-based principles. J Altern Complement Med. 2003; 9(3): 429–39. PubMed Abstract | Publisher Full Text\n\nFrass M, Strassl RP, Friehs H, et al.: Use and acceptance of complementary and alternative medicine among the general population and medical personnel: a systematic review. Ochsner J. 2012; 12(1): 45–56. PubMed Abstract | Free Full Text\n\nFreymann H, Rennie T, Bates I, et al.: Knowledge and use of complementary and alternative medicine among British undergraduate pharmacy students. Pharm World Sci. 2006; 28(1): 13–8. PubMed Abstract | Publisher Full Text\n\nGardiner P, Legedza A, Woods C, et al.: Herb use among health care professionals enrolled in an online curriculum on herbs and dietary supplements. J Herb Pharmacother. 2006; 6(2): 51–64. PubMed Abstract | Publisher Full Text\n\nGeldenhuys WJ, Cudnik ML, Krinsky DL, et al.: Evolution of a Natural Products and Nutraceuticals Course in the Pharmacy Curriculum. Am J Pharm Educ. 2015; 79(6): 82. PubMed Abstract | Free Full Text\n\nGyasi RM, Agyemang-Duah W, Mensah CM, et al.: Unconventional medical practices among Ghanaian students: A university-based survey. J Tradit Complement Med. 2016; 7(1): 126–132. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHina Y, Sana G, Fakhsheena A, et al.: Knowledge of herbal drugs among Pharm D. students in Karachi, Pakistan. Intl J Curr Pharm Rev Res. 2015; 6(4): 208–211. Reference Source\n\nJames PB, Bah AJ: Awareness, use, attitude and perceived need for Complementary and Alternative Medicine (CAM) education among undergraduate pharmacy students in Sierra Leone: a descriptive cross-sectional survey. BMC Complement Altern Med. 2014; 14: 438. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJamshed SQ, Khan MU, Ahmad A, et al.: Knowledge, perceptions, and attitudes toward complementary and alternative medicines among pharmacy students of a Malaysian Public University. J Pharm Bioallied Sci. 2016; 8(1): 34–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson SK, Blanchard A: Alternative medicine and herbal use among university students. J Am Coll Health. 2006; 55(3): 163–8. PubMed Abstract | Publisher Full Text\n\nJohnson T, Boon H, Jurgens T, et al.: Canadian pharmacy students' knowledge of herbal medicine. Am J Pharm Educ. 2008; 72(4): 75. PubMed Abstract | Free Full Text\n\nKarpa K: Development and implementation of an herbal and natural product elective in undergraduate medical education. BMC Complement Altern Med. 2012; 12: 57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKemper KJ, Gardiner P, Gobble J, et al.: Expertise about herbs and dietary supplements among diverse health professionals. BMC Complement Altern Med. 2006; 6: 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKilic S, Ogur R, Yaren H, et al.: Knowledge of and attitudes toward complementary and alternative medicine amongst medical students in a Turkish medical school. Pak J Med Sci. 2009; 25(2): 319–324. Reference Source\n\nKrenzelok EP, Mrvos R: Friends and foes in the plant world: a profile of plant ingestions and fatalities. Clin Toxicol (Phila). 2011; 49(3): 142–9. PubMed Abstract | Publisher Full Text\n\nLapidus M: Educating student pharmacists about herbal medicines: faculty-librarian collaboration. Health Info Libr J. 2007; 24(4): 267–73. PubMed Abstract | Publisher Full Text\n\nLarrañeta E: Incorporating Stories of Sedatives, Spoiled Sweet Clover Hay, and Plants from the Amazon Rainforest into a Pharmaceutical Chemistry Course to Engage Students and Introduce Drug Design Strategies. J Chem Educ. 2018; 95(10): 1778–1786. Publisher Full Text\n\nLee MS, Lim HJ, Lee MS, et al.: Perceptions, knowledge and misuse of an oriental herbal drug: a survey of 608 Korean female nursing college students. Complement Ther Clin Pract. 2005; 11(3): 200–4. PubMed Abstract | Publisher Full Text\n\nMackowiak ED, Parikh A, Freely J: Herbal product education in United States pharmacy schools: Core or elective program? Am J Pharm Educ. 2001; 65(1): 1–6. Reference Source\n\nMarcus DM, McCullough L: An evaluation of the evidence in \"evidence-based\" integrative medicine programs. Acad Med. 2009; 84(9): 1229–34. PubMed Abstract | Publisher Full Text\n\nMcKennon SA, Schauerhamer MB, Fudin HR, et al.: Assessing pharmacy student confidence to answer patient questions regarding herbal medicines and natural product drugs. Currents Pharm Teach Learn. 2018; 10(5): 643–650. PubMed Abstract | Publisher Full Text\n\nMei-Ying C, Huey-Shyan L, Chin-Fan T: Student nurses' knowledge, attitude, and behavior toward chinese medicine and related factors. J Nurs Res. 2004; 12(2): 103–18. PubMed Abstract | Publisher Full Text\n\nNg WY, Hung LY, Lam YH, et al.: Poisoning by toxic plants in Hong Kong: a 15-year review. Hong Kong Med J. 2019; 25(2): 102–112. PubMed Abstract | Publisher Full Text\n\nNworu CS, Udeogaranya PO, Okafor CK, et al.: Perception, usage and knowledge of herbal medicines by students and academic staff of University of Nigeria: A survey. Eur J Integr Med. 2015; 7(3): 218–227. Publisher Full Text\n\nOliveira SG, de Moura FR, Demarco FF, et al.: An ethnomedicinal survey on phytotherapy with professionals and patients from Basic Care Units in the Brazilian Unified Health System. J Ethnopharmacol. 2012; 140(2): 428–37. PubMed Abstract | Publisher Full Text\n\nOtieno NE, Analo C: Local indigenous knowledge about some medicinal plants in and around Kakamega forest in western Kenya [version 2; peer review: 2 approved]. F1000Research. 2012; 1: 40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOwen DJ, Fang ML: Information-seeking behavior in complementary and alternative medicine (CAM): an online survey of faculty at a health sciences campus. J Med Libr Assoc. 2003; 91(3): 311–21. PubMed Abstract | Free Full Text\n\nPan JH, Peng CY, Lo CT, et al.: n-Hexane intoxication in a Chinese medicine pharmaceutical plant: a case report. J Med Case Rep. 2017; 11(1): 120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaha BL, Seam MOR, Islam MM, et al.: General perception and self-practice of complementary and alternative medicine (CAM) among undergraduate pharmacy students of Bangladesh. BMC Complement Altern Med. 2017; 17(1): 314. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSamara AM, Barabra ER, Quzaih HN, et al.: Use and acceptance of complementary and alternative medicine among medical students: a cross sectional study from Palestine. BMC Complement Altern Med. 2019; 19(1): 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSawalha AF, Sweileh WM, Zyoud SH, et al.: Self-therapy practices among university students in Palestine: focus on herbal remedies. Complement Ther Med. 2008; 16(6): 343–9. PubMed Abstract | Publisher Full Text\n\nSiti ZM, Tahir A, Farah AI, et al.: Use of traditional and complementary medicine in Malaysia: a baseline study. Complement Ther Med. 2009; 17(5–6): 292–9. PubMed Abstract | Publisher Full Text\n\nSoroush A, Abdi A, Andayeshgar B, et al.: Exploring the perceived factors that affect self-medication among nursing students: a qualitative study. BMC Nurs. 2018; 17: 35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSriapha C, Tongpoo A, Wongvisavakorn S, et al.: Plant Poisoning in Thailand: A 10-Year Analysis From Ramathibodi Poison Center. Southeast Asian J Trop Med Public Health. 2015; 46(6): 1063–76. PubMed Abstract\n\nSuchard JR, Suchard MA, Steinfeldt JL: Physician knowledge of herbal toxicities and adverse herb-drug interactions. Eur J Emerg Med. 2004; 11(4): 193–7. PubMed Abstract | Publisher Full Text\n\nXu S, Levine M: Medical residents' and students' attitudes towards herbal medicines: a pilot study. Can J Clin Pharmacol. 2008; 15(1): e1–4. PubMed Abstract\n\nZamawe C, King C, Jennings HM, et al.: Associations between the use of herbal medicines and adverse pregnancy outcomes in rural Malawi: a secondary analysis of randomised controlled trial data. BMC Complement Altern Med. 2018; 18(1): 166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimmerman C, Kandiah J: A pilot study to assess students' perceptions, familiarity, and knowledge in the use of complementary and alternative herbal supplements in health promotion. Altern Ther Health Med. 2012; 18(5): 28–33. PubMed Abstract"
}
|
[
{
"id": "69639",
"date": "24 Sep 2020",
"name": "Venil Sumantran",
"expertise": [
"Reviewer Expertise Cell and Cancer Biology",
"Biochemistry",
"Pre-clinical studies",
"Alternative Medicine (Ayurveda)"
],
"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\nReported findings may be correct and valid, but the paper is 'Not Approved' because study design and data analysis are weak.\n\nStudy design:\n\nSince there is no control group—the sample size should have been >50.\n\nDid the subjects use herbal drugs along with standard (allopathic) drugs, lifestyle changes, i.e. as a complementary mode of treatment, or were these herbal drugs the sole mode of treatment (Alternative) for a given ailment?\n\nThe word ‘cure’ isn’t defined. Figure 5 shows that 3 of the 6 ailments involve pain. So, a validated pain score could have been used.\n\nData analysis:\n\nThe Authors mention using different statistical tests, but only Figure 2 has a p-value.\n\nThe Authors do not clearly mention the “cure rate\". The data and text imply that 95.80 % were cured and 4% were not.\n\nThe very high cure rate (96%) could be due to a bias arising from recruiting students who strongly lean towards herbal medicine, and points to a placebo effect. Even though this point is not the focus of their study, authors should discuss reasons for the high ‘cure rate’-purely from viewpoint of thorough data analysis.\n\nSince the hypothesis states that health professionals should have greater knowledge about these herbs than laymen, a Chi-squared test for association would show if the knowledge source was significantly associated with the major subject studied by students. The Chi-squared test could compare Knowledge Source ( Research, Social media, Others) versus students of Biomed Sci + Biol Sci (20) and students of Nursing +Pharmacy (30). Why was this not done?\n\nFigure 6 shows the 3 most commonly used herbs in the study. There is no analysis. The Authors could make a Pie chart/Table for each of the 3 herbs. Four parameters could be considered. (1. Herb taken for correct ailment 2. Herb taken for incorrect ailment 3. Expected outcome 4. Actual outcome). Based on these data, additional Chi-squared tests may be required.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "92072",
"date": "02 Sep 2021",
"name": "Alan Joel Ruiz-Padilla",
"expertise": [
"Reviewer Expertise Pharmaceutical care",
"Pharmaceutical Care Services",
"Self-medication",
"Epidemiology",
"Natural Products."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe subject and objective are interesting, worth investigating. However, the study lacks a clear and concrete methodological design. There are inconsistencies between methodology, results, and conclusions.\nStudy design\nThe sample size is too small for a descriptive cross-sectional study.\n\nThe population studied is not homogeneous and it is not disputed whether these factors could have affected its results.\na. Knowledge of plant use and research through scientific information resources may differ between beginner and advanced grade students.\n\nb. The differences in proportions of the sex of the participants.\n\nThere are several factors not included that could intervene with the results of the consumption of medicinal plants, such as the mode, dose, and manner of consumption of medicinal plants, presence of comorbidities, and other medications.\n\nIn the survey carried out, when they ask who indicated the use of these medicinal plants, they do not offer the possibility that their consumption has been by a health professional indication, they are predetermining that it is self-consumption. This could explain the high percentages of self-consumption in this group of people.\nResults\nFigure 3. The meaning of the areas (black and white) of the graphs is not described.\n\nThey equate the concept of “curing diseases” with that of “obtaining some positive effect”. These concepts are only the authors' interpretation, and they are not supported by the results of the survey since the question only addresses compliance with the expectations of the respondent.\n\nFigures 4 and 6. It is not specified what the Y-axis represents in the bar graph.\n\nIt would be more interesting if results were presented on the use of medicinal plants for specific symptoms, as well as what was their main source of recommendation.\nDiscussion and Conclusions\nAn important part of the discussion involves physicians; however, students of this profession were not included in the study population, their inclusion would be very useful.\n\nThe survey only allows to choose Sources not based on evidence, participants do not have the option to select Sources of Information Based on evidence; therefore the discussion on this point is meaningless.\n\nThe lack of desire to answer the survey is described as a limitation, it will help to know the proportion of acceptance of participation.\n\nIt is appropriate to point out that these results cannot be generalized.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-244
|
https://f1000research.com/articles/9-20/v1
|
14 Jan 20
|
{
"type": "Brief Report",
"title": "The effects of microchipping C57BL/6N mice on standard phenotyping tests",
"authors": [
"R.S. Bains",
"H.L. Cater",
"M. Stewart",
"C.L. Scudamore",
"S.E. Wells",
"H.L. Cater",
"M. Stewart",
"C.L. Scudamore",
"S.E. Wells"
],
"abstract": "The C57BL/6N inbred lines of mice are widely used in genetic research. They are particularly favoured in large scale studies such as the International Mouse Phenotyping Consortium (IMPC), where C57BL/6N mice are genetically altered to generate a collection of null alleles (currently more than 8500 null alleles have been generated). In this project, mice carrying null alleles are subjected to a pipeline of broad-based phenotyping tests to produce wide ranging phenotyping data on each model. We have previously described the development of a Home Cage Analysis system that automatically tracks the activity of group housed mice from a microchip inserted in the groin. This platform allows assessment of multiple biologically relevant phenotypes over long periods of time without experimenter interference, and therefore is particularly suited for high through-put studies. To investigate the impact of microchips on other tests carried out in the IMPC pipeline, we inserted microchips in 12 male and 12 female C57BL/6Ntac mice at seven weeks of age. Starting at nine weeks of age these mice underwent standard phenotyping tests, concurrently with 20 unchipped C57BL/6Ntac mice (10 females, 10 males). Tissues from a subset of the microchipped mice (six males and six females), chosen at random, were also sent for histopathological examination at the end of the phenotyping pipeline.",
"keywords": [
"RFID",
"Microchips",
"phenotyping",
"C57BL/6NTac",
"mice",
"Home cage analysis",
"histopathology"
],
"content": "Introduction\n\nThe overarching aim of phenotyping studies is to understand and describe the relationship between genotype and phenotype. However, mouse behaviour is variable, dynamic and adaptive and therefore is influenced by a variety of genetic and environmental factors such as motivation, interaction with the experimenter, experimental design, test order, testing time and environmental enrichment1,2. In recent years, automated home cage phenotyping as a complement to conventional out-of-cage testing has increasingly been used to enrich phenotype datasets3. We have been developing a novel Home Cage Analysis system (HCA) for multiply housed mice that is entirely compatible with the modern high density individually ventilated caging (IVC) systems. The HCA allows us to record automated detailed behavioural parameters over time in an undisturbed standard rack-mounted cage - the type the mice are born, reared and constantly housed in - within their established social groups. The HCA combines radio frequency identifier (RFID) tracking with video recording for automated behavioural scoring. For optimal resolution of the RFID signals from the microchips, the chips are inserted subcutaneously in the animal’s groin4. Though designed to investigate subtle phenotypes in undisturbed animals, thereby reducing variability, it is possible that the insertion of the RFID microchip may itself influence behaviour, thus changing the phenotyping profile.\n\nImplantable micro-identification has been used as an effective means of identification in long term rodent studies for over two decades; where a hermetically sealed inert glass cylinder, 12mm in length and 2mm in diameter, containing the microchip transponder is implanted subcutaneously in the mid dorsal region of the animal5,6. Although widely considered safe, some long term studies have reported the development of foreign-body-induced tumours in various mouse strains7,8. Furthermore, the subcutaneous implantation of the microchip in the groin requires the mice to be anaesthetised; although gaseous anaesthesia is brief (less than one minute), this may interfere with the phenotyping tests further downstream9. The implant itself may also confound certain assays such as dual-energy X-ray absorptiometry (DEXA) which measures total body composition. The current study investigates phenotypic effects of subcutaneous microchip implants in the groin of C57BL/6NTac mice. At the end of the study the tissue around the microchip was sent for histopathology to detect any signs of foreign-body induced pathology.\n\n\nMethods\n\nAll procedures and animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986, UK, Amendment Regulations 2012 (SI 4 2012/3039). This study has been approved by the Animal Welfare and Ethical Review Board (AWERB), resulting in licence 30/2890. All efforts were made to minimize suffering by considerate housing and husbandry, the details of which are available at the IMPC portal: http://www.mousephenotype.org/about-impc/arrive-guidelines. Animal welfare was assessed routinely for all mice involved.\n\nAdult mice were killed by terminal anaesthesia followed by exsanguination, and death was confirmed by either cervical dislocation or cessation of circulation.\n\n22 C57BL/6NTac male and 22 C57BL/6NTac female mice, bred at the Mary Lyon Centre, Harwell, were housed in IVC’s, on Eco-pure spruce chips grade 6 bedding (Datesand, U.K.), with shredded paper shaving nesting material and small cardboard play tunnels for enrichment. Mice were randomly assigned to groups of three to five mice per cage and the phenotyping tests were carried out as a part of a bigger batch of experimental animals and their position in the batch was randomly allocated. While the method of randomisation was simple, the experimenters were blinded to the test groups. Only the experimenters carrying out X-ray analysis after the test would know the groups as that would not be possible to blind. The mice were kept under controlled light (light 7 a.m. to 7 p.m., dark 7 p.m. to 7 a.m.), temperature (21 °C ± 2 °C) and humidity (55% ± 10%) conditions. They had free access to water (25 p.p.m. chlorine) and were fed ad libitum on a commercial diet (SDS Rat and Mouse No.3 Breeding diet (RM3)).\n\nAnimal welfare checks were carried out visually once a day.\n\nThe microchipping procedure has been described in Bains et al., 20164. Briefly, RFID microchips were injected subcutaneously into the lower left or right quadrant of the abdomen of each mouse at seven weeks of age. These microchips were contained in standard ISO biocompatible glass capsules (11.5×2mm, PeddyMark Ltd. UK). The procedure was performed on sedated mice (Isoflo, Abbott, UK) after topical application of local anaesthetic cream on the injection site prior to the procedure (EMLA Cream 5%, AstraZeneca, UK). While using local anaesthetic is not common practice, we have found that a combination of these anaesthetics is the most refined method of delivering these scientific data, as this minimizes any acute discomfort the animal may feel after recovering from the general anaesthetic.\n\nNo sutures were required.\n\n12 male and 12 female C57BL/6NTac mice were microchipped in the groin at seven weeks of age and phenotyped alongside 10 male and 10 female C57BL/6NTac baseline mice (n=24-20), starting at nine weeks of age. 20 baseline and 24 microchipped mice underwent systematic broad-based phenotyping for adults, according to the pipeline described in Table 1. The reason for the difference in numbers in each experimental group was due to the HCA being optimised for three animals in a home cage while the mice in the standard phenotyping pipeline are housed in groups of five. A sample size of 10 mice per sex was calculated based on an alpha of 1% and power of 0.8. All standard operating procedures have been described within the International Mouse Phenotyping Resource of Standardised Screens, the IMPReSS database (www.mousephenotype.org/impress).\n\nThe table outlines the phenotyping tests in the pipeline in the order that they are carried out in, along with the age in weeks that each test is conducted.\n\nSHIRPA, SmithKline Beecham, Harwell, Imperial College and Royal London Hospital phenotype assessment; IPGTT, intraperitoneal glucose tolerance test; DEXA, dual energy X-ray analysis.\n\nTo provide traceability, IMPReSS also stores a record of change histories, all tests in this study were carried out at the Mary Lyon Centre, MRC Harwell Institute, between November 2015 and March 2016.\n\nAt the end of the phenotyping pipeline, six randomly chosen microchipped males and six randomly chosen microchipped females at 16 weeks of age underwent necropsy. The animals were simultaneously randomised without considering any variable as a part of a larger batch of animals undergoing the same procedure. The choice of euthanasia, overdose of anaesthetic followed by exsanguination, was considered the most refined and appropriate for gathering the data for clinical chemistry and haematology assays by the AWERB. The site of microchip implantation was examined and the tissue surrounding the implant was fixed in 10% neutral buffered formalin (NBF). Tissue samples from the implant sites were processed routinely to wax and 3µm sections were stained with haematoxylin and eosin (H&E) and examined by a veterinary pathologist.\n\nData for Open Field test, Grip Strength test, Acoustic Startle and Pre-pulse Inhibition and DEXA were analysed using the Student’s t-test with Welch’s correction for unequal variance. The data for weekly weight curve and intraperitoneal glucose tolerance test (IPGTT) were assessed using two-way analysis of variance (ANOVA) with Sidak post hoc analysis. As the current study tests multiple hypotheses, Bonferroni’s correction was applied to avoid type 1 error in the interpretation of the data. Therefore, an alpha value of < 0.005 was calculated for an effect to have statistical significance. The categorical data such as Combined SHIRPA and Dysmorphology (CSD), opthalmology and X-ray were analysed using Fisher’s exact test. GraphPad Prism 8.3.0 was used for statistical analysis.\n\n\nResults\n\nDysmorphology. No evidence of any major differences in morphological features was found between the chipped and unchipped mice. The X-ray analysis showed that microchips were situated at the site of implantation.\n\nMetabolism. There was no significant difference between the weight curve data of chipped mice compared to unchipped mice over the 12-week testing period10. Figure 1C represents the mean body weight±sem of each group compared every week starting at four weeks of age (16.71±0.29 non-chipped vs 16.13±0.46 chipped females; 16.5±0.63 non-chipped vs 18.22±0.29 chipped males), until the end of the study at 16 weeks (24.22±0.27 non-chipped vs 25.1±0.5 chipped for females; 31.37±0.19 non-chipped vs 30.7±0.41 chipped males). DEXA body composition measurements, represented in Figure 1A, showed significant differences between the two groups, when analyzing bone mineral density (p<0.0001; mean±sem= 0.054±0.001 non-chipped vs 0.063±0.001 chipped females; 0.057±0.001 non-chipped vs 0.067±0.001 chipped males) and bone mineral content (p<0.001; 0.49±0.01 non-chipped vs 0.56±0.01 chipped females; 0.55±0.01 non-chipped vs 0.63±0.01 chipped males) in both males and females. This can be attributed to the texture of the microchip, which would behave similarly to bone when imaged using DEXA. The results from the IPGTT, represented in Figure 1B, showed an increase in mean blood glucose concentration at the 15-minute time point for microchipped females (30.73±1.87) versus unchipped females (23.88±1.04), but this was not statistically significant. No evidence of altered glucose metabolism was seen in chipped males (17.99±0.87) versus unchipped males (20.76±0.86) for the 15-minute time point.\n\nA) Bars represent average bone mineral density (g/cm2) and average bone mineral content (g), for males and females as measured through dual-energy X-ray absorptiometry (DEXA). Microchipped females as well as males show a significant increase in the two parameters as compared to unchipped animals (n=10-12; ***p<0.001 and ****p<0.0001), the T bars represent ±sem. B) The lines represent average glucose concentration (mmol/L) over time as measured through IPGTT, the shapes represent average glucose concentration (mmol/L) for each group at that time point, the T bars represent ±sem. No statistically significant difference was seen in either sex for microchipped versus unchipped mice. C) The lines represent the body weight curve of the two treatment groups over time, the shapes represent mean body weight (gm) for each group at that time point, the T bars represent ±sem. No statistically significant difference was seen in either sex for microchipped versus unchipped mice.\n\nNeurological, behavioral and sensory. No significant differences were observed in the whole arena open field locomotor parameters between chipped and unchipped mice (means±sem= 12.08±0.40 for non-chipped vs 12.32±0.36 chipped females; means±sem=12.06±0.51 for non-chipped vs 10.73±0.19 chipped for males); this was also true for the locomotor activity observed in the arena during the modified SHIRPA testing, where all animals tested showed normal activity. Grip strength measurements of fore and hind limbs normalized to body weights between the two groups were also non-significant (means±sem=4.89±0.19 for non-chipped vs 5.12±0.19 chipped females; means±sem=4.91±0.21 for non-chipped vs 4.83±0.24 chipped for males).\n\nAcoustic startle (means±sem of startle amplitude= 481.75±29.86 non-chipped vs 443.79±31.19 for females; 668.33±53.71 non-chipped vs 499.27±50.39 chipped males) and startle response to pre-pulse showed no significant difference between the chipped and unchipped mice (means±sem of pre pulse1 startle amplitude= 530.64±48.46 non-chipped vs 418.6±23.9 for females; 659.28±57.03 non-chipped vs 504.65±42.24 chipped males; means±sem of pre pulse2 startle amplitude= 298.03±19.76 non-chipped vs 254.7±18.96 for females; 315.35±32.48 non-chipped vs 275.58±32.29 chipped males; means±sem of pre pulse3 startle amplitude= 302.53±18.37 non-chipped vs 226.67±12.7 for females; 308.27±33.65 non-chipped vs 272.91±32.29 chipped males.\n\nDuring the lifespan of the mice, the general health of the mice was considered normal and no clinical symptoms associated with the implant sites were observed.\n\nOn microscopic examination of the implant sites, a thin fibrous capsule was identified in the subcutaneous tissue around a central space where the implant would have been located in 50% of the mice. In two of the 12 animals, small amounts of foreign material, with an associated minimal focal foreign body reaction, was identified in the adipose connective tissue adjacent to the fibrous capsule. These lesions were minor and considered to be of no clinical significance. There was no evidence of generalized or diffuse inflammation, uncontrolled mesenchymal proliferation or tumor formation.\n\n\nConclusion\n\nInsertion of microchips in the groin has no significant effect on the phenotype of mice when assessed using the standard IMPC phenotyping pipeline. While the DEXA results show a significant difference in bone mineral density and bone mineral content between non-chipped and chipped mice, this is to be expected. DEXA relies on transmission of X-rays with high and low energies through a medium to determine the mass per unit (or density) of the medium11; RFID microchips in this scenario being a dense hard material behave as bone and skew the results. The microchip therefore affects the results of this test but not the actual bone mineral content or bone mineral density of the mice. However, if microchips are implanted in both test groups and controls this should have no overall effect on the study. The implant does not appear to have any adverse welfare effects on the health of the mice.\n\nThe phenotype of an organism is an indicator of how it will function in different environments and under different challenges. Recording the phenotype of a mouse constitutes recording every aspect of clinical, morphological, physiological or cellular change that may arise from any type of experimental manipulation: genetic, surgical or pharmacological12,13.\n\nInsertion of microchips in the groin requires the application of general anaesthesia; the post-anaesthetic effects of isoflurane on learning and memory may affect behaviour phenotyping9,14–16, although this is unlikely with such brief anaesthesia. The current study does not detect any noticeable effects on the phenotype of the mice. However, the data is limited to a single inbred wild type strain; genetically modified mice with unknown phenotypes may react differently to the required exposure to anaesthesia.\n\n\nData availability\n\nHarvard Dataverse: The effects of microchipping C57BL/6N mice on standard phenotyping tests. https://doi.org/10.7910/DVN/PFLBYU10\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\nLoos M, Koopmans B, Aarts E, et al.: Sheltering behavior and locomotor activity in 11 genetically diverse common inbred mouse strains using home-cage monitoring. PLoS One. 2014; 9(9): e108563. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHong W, Kennedy A, Burgos-Artizzu XP, et al.: Automated measurement of mouse social behaviors using depth sensing, video tracking, and machine learning. Proc Natl Acad Sci U S A. 2015; 112(38): E5351–E5360. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBains RS, Wells S, Sillito RR, et al.: Assessing mouse behaviour throughout the light/dark cycle using automated in-cage analysis tools. J Neurosci Methods. 2018; 300: 37–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBains RS, Cater HL, Sillito RR, et al.: Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System. Front Behav Neurosci. 2016; 10: 106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRao GN, Edmondson J: Tissue reaction to an implantable identification device in mice. Toxicol Pathol. 1990; 18(3): 412–416. PubMed Abstract | Publisher Full Text\n\nLe Calvez S, Perron-Lepage MF, Burnett R: Subcutaneous microchip-associated tumours in B6C3F1 mice: A retrospective study to attempt to determine their histogenesis. Exp Toxicol Pathol. 2006; 57(4): 255–265. PubMed Abstract | Publisher Full Text\n\nTillmann T, Kamino K, Dasenbrock C, et al.: Subcutaneous soft tissue tumours at the site of implanted microchips in mice. Exp Toxicol Pathol. 1997; 49(3–4): 197–200. PubMed Abstract | Publisher Full Text\n\nLapp S, Bube A, Colbatzky FA, et al.: Best Practice Approach for Assessment of Microchip-associated Tumors in Preclinical Safety Studies: Position of the Registry of Industrial Toxicology Animal-data (RITA). Toxicol Pathol. 2018; 46(7): 728–734. PubMed Abstract | Publisher Full Text\n\nYonezaki K, Uchimoto K, Miyazaki T, et al.: Postanesthetic effects of isoflurane on behavioral phenotypes of adult male C57BL/6J mice. PLoS One. 2015; 10(3): e012211823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBains RS: \"The effects of microchipping C57BL/6N mice on standard phenotyping tests\". Harvard Dataverse, V1, UNF:6:NIHxnYrJ5ZYje5NdBH76Sw== [fileUNF], 2019. http://www.doi.org/10.7910/DVN/PFLBYU\n\nBlake GM, Fogelman I: Technical principles of dual energy x-ray absorptiometry. Semin Nucl Med. 1997; 27(3): 210–228. PubMed Abstract | Publisher Full Text\n\nBrown SD, Hancock JM, Gates H: Understanding mammalian genetic systems: the challenge of phenotyping in the mouse. PLoS Genet. 2006; 2(8): e118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeiss CJ, Ward JM, Allore HG: Designing phenotyping studies for genetically engineered mice. Vet Pathol. 2012; 49(1): 24–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCao L, Li L, Lin D, et al.: Isoflurane induces learning impairment that is mediated by interleukin 1β in rodents. PLoS One. 2012; 7(12): e51431. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFidalgo AR, Cibelli M, White JP, et al.: Isoflurane causes neocortical but not hippocampal-dependent memory impairment in mice. Acta Anaesthesiol Scand. 2012; 56(8): 1052–1057. PubMed Abstract | Publisher Full Text\n\nLee S, Park SH, Zuo Z: Effects of isoflurane on learning and memory functions of wild-type and glutamate transporter type 3 knockout mice. J Pharm Pharmacol. 2012; 64(2): 302–307. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "58610",
"date": "23 Jan 2020",
"name": "Jonathan M. Williams",
"expertise": [
"Reviewer Expertise Veterinary anatomic pathology and intestinal 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\nIn this brief report manuscript, entitled “The effects of microchipping C57BL/6N mice on standard phenotyping tests”, Bains et al. describe the development of a home cage analysis system which tracks activity mouse activity through a RFID microchip placed in the groin in 12 male and 12 female c57bl/6 mice at 7 weeks of age.\nThis is of particular value as RFID microchips can measure a large number of phenotypic responses with frequent measurements over large time periods without handling or stress inducement in the animal.\nThe results demonstrate that importantly, the procedure of chip implantation does not significantly alter across a large number of measurable phenotypic responses - only causing significant changes in measurements of bone density.\nOverall this is a well written report.\nI have only minor criticisms relating to the methods:\nIn the husbandry section, it is stated that “While the method of randomisation was simple…”- it is not described. What was the method of randomisation?\n\nAre animals sedated or under general anaesthesia for chip insertion? Under the microchipping section, it states “This procedure was performed on sedated mice” but elsewhere in the manuscript general anaesthesia is frequently referenced.\n\nIt also states “we have found that a combination of these anaesthetics is the most refined method of delivering these scientific data, as this minimizes any acute discomfort the animal may feel after recovering from the general anaesthetic.”\n\nThere are no details of the manufacturer/source of the RFID chip or software used which would present problems in attempting replication of 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? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5191",
"date": "04 Feb 2020",
"name": "Rasneer Sonia Bains",
"role": "Author Response",
"response": "We would like to thank the reviewer for taking the time to review our manuscript and for sharing their comments. Please find below our response to the same. 1. In the husbandry section, it is stated that “While the method of randomisation was simple…”- it is not described. What was the method of randomisation? Response: The method of randomisation was ‘simple randomisation’, described in the arrive guidelines as, “All animals/samples are simultaneously randomised to the treatment groups without considering any other variable. This strategy is rarely appropriate as it cannot ensure that comparison groups are balanced for factors or covariates that might influence the result of an experiment.” We will clarify that in the text by rewording the statement to “The method of randomisation used was Simple Randomisation…” 2. Are animals sedated or under general anaesthesia for chip insertion? Under the microchipping section, it states “This procedure was performed on sedated mice” but elsewhere in the manuscript general anaesthesia is frequently referenced.It also states “we have found that a combination of these anaesthetics is the most refined method of delivering these scientific data, as this minimizes any acute discomfort the animal may feel after recovering from the general anaesthetic.” Response: The procedure was performed on anaesthetised mice, we will change the text in this section to reflect that. 3. There are no details of the manufacturer/source of the RFID chip or software used which would present problems in attempting replication of this experiment. Response: The details of the RFID microchips are supplied in the Microchipping section of the manuscript, “These microchips were contained in standard ISO biocompatible glass capsules (11.5×2mm, PeddyMark Ltd. UK).”The software used to read these RFID data is described in reference 4 - Bains et al., 2016, where the details of the Home Cage Analysis system (Actual Analytics, Edinburgh) have been published. As the RFID data were not part of the current manuscript, the details of the software were not considered essential to the replicate this study."
}
]
},
{
"id": "58613",
"date": "04 Feb 2020",
"name": "Helmut Fuchs",
"expertise": [
"Reviewer Expertise Mouse phenotyping"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “The effects of microchipping C57BL/6N mice on standard phenotyping tests” by Bains et al. describes the comparison of phenotyping data from 12 male and 12 female mice that were microchipped in the groin with unchipped animals (10 males and 10 females). The mice were wild-type C57BL/6N and were analyzed in the IMPC phenotyping pipeline. The experiment is highly interesting, as in the mouse phenotyping community there is a lot of effort put in the development of home cage monitoring systems for an undisturbed observation and analysis of mouse behavior in their normal environment. State of the art technologies require microchipping of the mice for a proper identification of individual mice in the system. Thus, it is of importance to investigate the impact of microchips on other tests that are carried out within the IMPC phenotyping pipeline.\nThe experiments and results are well described in the manuscript, however, there remain some questions about the selected methodology and the possible conclusions: we are missing a clear conclusion in the abstract. In the conclusion part there are three statements: “Insertion of microchips in the groin has no significant effect on the phenotype of mice when assessed using the standard IMPC phenotyping pipeline”. “The implant does not appear to have any adverse welfare effects on the health of the mice”. The third conclusion is “The current study does not detect any noticeable effects on the phenotype of the mice”. How far is it via the selected approach possible to obtain information about effects of microchipping on other phenotyping tests – or in other words: how probable is it that there are really no differences if under the given conditions and analysis no differences were detected? For example, the neurological, behavioral and sensory analysis data: The authors decided for an alpha value of 1% and in addition did a correction for multiple testing. Thus, the observed differences need to be very high to reach statistical significance.\n\nAdditional comments:\nStatistical analysis: On which parameters and which tests was the sample size and power calculation based? More detailed information on the power calculation would be desirable. Is there a special reason why the authors select an alpha value of 1%? Also, a more detailed description on the method for multiple testing correction would be helpful.\n\nMaybe the results part should reflect the order of the tests in the phenotyping pipeline. For a couple of tests from the mentioned phenotyping pipeline there are no results mentioned. For example, in the open-field and SHIRPA analysis the authors report only data from locomotion related parameters. What about the other areas that are covered by these tests (e.g. anxiety and exploratory behaviors)?\n\nBody weight: According to the provided graphs, at the age of chip implantation (7 weeks) the body weight data flips. In females, non-chipped mice are below, and afterwards are heavier than chipped mice. In males, non-chipped mice are below the chipped mice and then are heavier. Is there any idea about the reason? What is the weight of the RFID? Can the weight of the chip itself make a difference in body weight? It might be interesting to know whether the authors also considered other types of RFID’s for the experiment, and why they selected the used one for the study?\n\nDEXA analysis: Is it possible to cut out the area that is covered by the chip from the DEXA image for the analysis? A DEXA-Image of a mouse with chip compared with an unchipped mouse might be helpful. Is there more detailed information available about the material contained inside the chip? If it contains some metal, it might cause scattered radiation that could be harmful for the mouse, and if the DEXA-system is not completely shielded (as it might be the case for older DEXA-systems), also for the experimenter.\n\nIs the work 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5307",
"date": "16 Mar 2020",
"name": "Rasneer Sonia Bains",
"role": "Author Response",
"response": "Response: We thank the reviewers for their comments and suggestions on our manuscript. We have tried to address the concerns raised in the responses below and would be amending the manuscript to reflect these changes. The manuscript “The effects of microchipping C57BL/6N mice on standard phenotyping tests” by Bains et al. describes the comparison of phenotyping data from 12 male and 12 female mice that were microchipped in the groin with unchipped animals (10 males and 10 females). The mice were wild-type C57BL/6N and were analyzed in the IMPC phenotyping pipeline. The experiment is highly interesting, as in the mouse phenotyping community there is a lot of effort put in the development of home cage monitoring systems for an undisturbed observation and analysis of mouse behavior in their normal environment. State of the art technologies require microchipping of the mice for a proper identification of individual mice in the system. Thus, it is of importance to investigate the impact of microchips on other tests that are carried out within the IMPC phenotyping pipeline.The experiments and results are well described in the manuscript, however, there remain some questions about the selected methodology and the possible conclusions: we are missing a clear conclusion in the abstract.Response: The authors will add the following text in the abstract: “No significant impact of insertion of microchip was observed in any of the phenotyping tests apart from bone mineral density measurement at DEXA due to the nature of the microchip. We therefore recommend that the microchip be inserted during the DEXA procedure, after the measurement is taken but before the mouse has recovered from the anaesthetic. This would avoid multiple anaesthetic exposures and prevent the potential variability in DEXA analysis output.”In the conclusion part there are three statements: “Insertion of microchips in the groin has no significant effect on the phenotype of mice when assessed using the standard IMPC phenotyping pipeline”. “The implant does not appear to have any adverse welfare effects on the health of the mice”. The third conclusion is “The current study does not detect any noticeable effects on the phenotype of the mice”. How far is it via the selected approach possible to obtain information about effects of microchipping on other phenotyping tests – or in other words: how probable is it that there are really no differences if under the given conditions and analysis no differences were detected? For example, the neurological, behavioral and sensory analysis data: The authors decided for an alpha value of 1% and in addition did a correction for multiple testing. Thus, the observed differences need to be very high to reach statistical significance.Response: We agree with the concerns raised by the reviewers about the stringent criteria, however, the aim of the current study was to establish the effect of a single procedure on a whole pipeline. We therefore decided to choose the middle ground between using an alpha of 5% and 0.01%, without compromising the integrity of the study. Additional comments: Statistical analysis: On which parameters and which tests was the sample size and power calculation based? More detailed information on the power calculation would be desirable. Is there a special reason why the authors select an alpha value of 1%? Also, a more detailed description on the method for multiple testing correction would be helpful. Response: As discussed in Karp et al., 2015, the calculation of false discovery rate is an ongoing discussion within the consortia. While the current work draws from the ARRIVE guidelines published in Karp et al., 2015, it was felt that as the current study was only carried out in one centre and over a short period of time using an alpha of 0.0001 would perhaps be too stringent and lead to a type 2 error. We therefore chose an alpha value of 1%. Based on an alpha of 0.01 and R=11 tests the alpha was calculated to be 0.001, and not 0.005 as initially reported in the manuscript. We thank the reviewers for picking up this typo, we would like to add here that this does not alter the results. We will correct the text to reflect this. Maybe the results part should reflect the order of the tests in the phenotyping pipeline. For a couple of tests from the mentioned phenotyping pipeline there are no results mentioned. For example, in the open-field and SHIRPA analysis the authors report only data from locomotion related parameters. What about the other areas that are covered by these tests (e.g. anxiety and exploratory behaviors)? Response: It was not possible, given the character limit to include every parameter in the main body of the text, however all data points have been included in the data repository. Locomotor activity was chosen as the representative parameter as this was the most likely aspect of the mouse behaviour to be affected by insertion of a microchip to the groin. The other biological system most likely to be effected was the metabolism and as such was included in the main body of the text. Body weight: According to the provided graphs, at the age of chip implantation (7 weeks) the body weight data flips. In females, non-chipped mice are below, and afterwards are heavier than chipped mice. In males, non-chipped mice are below the chipped mice and then are heavier. Is there any idea about the reason? What is the weight of the RFID? Can the weight of the chip itself make a difference in body weight?It might be interesting to know whether the authors also considered other types of RFID’s for the experiment, and why they selected the used one for the study? Response: The weights when expressed as individual data points show a near complete overlap and there is no difference in the two groups in either sex. The slight changes in trend can be attributed to individual animals that have a low in case of females and high starting weight in case of males, which were then randomly assigned to one of the two groups and have therefore continued to influence the trend through the study period.The RfiDs used in the current study on average weigh approximate 100mg, it is not currently possible to use a wide variety of microchips from different manufacturers as the baseplate readers are tuned to particular chip types and it is not simply a case of swapping one chip for another without tuning and validating the new chips. As the chips used in the current study were used to develop and validate the system we did not deem it necessary to change the chips for the current study. DEXA analysis: Is it possible to cut out the area that is covered by the chip from the DEXA image for the analysis? A DEXA-Image of a mouse with chip compared with an unchipped mouse might be helpful.Is there more detailed information available about the material contained inside the chip? If it contains some metal, it might cause scattered radiation that could be harmful for the mouse, and if the DEXA-system is not completely shielded (as it might be the case for older DEXA-systems), also for the experimenter. Response: It is possible to analyse the data as suggested, and we will include this suggestion in our conclusion. In addition, we will also include the images as suggested in the supplementary data with the paper.While the equipment in our laboratories is well shielded and the harm from scattered radiation is very small. The legislation in the U.K. has approved the microchips for use in pets. We agree with the concerns raised and therefore recommend that the mice be chipped after DEXA, before the mice recover from anaesthetic. We will include this in our manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/9-20
|
https://f1000research.com/articles/9-95/v1
|
07 Feb 20
|
{
"type": "Case Report",
"title": "Case Report: Rare site for intraoral meningioma",
"authors": [
"Hatem Wael Amer",
"Layla Hafed",
"Sally Ibrahim",
"Shady Shaker",
"Hatem Wael Amer",
"Sally Ibrahim",
"Shady Shaker"
],
"abstract": "Extracranial meningioma is very rare with few cases reported, especially in the oral cavity. Its diagnosis considered a challenge owing to the unusual site of occurrence. We report, to our knowledge, the first case of extra-cranial meningioma as a primary tumor in the palate with no detected intracranial extension. A 59-year-old female Egyptian patient presented with a 22-year history of a large painless swelling at the right side of the palate, which could not be seen on radiographs. An incisional biopsy was taken and, after assessment with a panel of immunohistochemical markers, the lesion was diagnosed as extracranical meningioma. The patient did not show up for surgical excision and follow-up was not performed because of loose of contact with the patient. Intraoral meningioma is a rare unsuspected tumor. Immuohistochemical markers are important when confirming this diagnosis.",
"keywords": [
"Intra-oral meningioma",
"Benign tumor",
"Ectopic meningioma",
"Palatal lesion"
],
"content": "Introduction\n\nMeningioma is a benign neoplasm of meningothelial cells1. Meningioma may develop as a direct extension of a primary intra-cranial meningioma or as a true primary extra-cranial meningioma2.\n\nExtra-cranial (ectopic) tumors are mostly seen in the head and neck region with no connection intra-cranially3. The most common extra-cranial site is the orbits. Meningioma arising in the oral cavity is extremely rare4. To the extent of our knowledge, 18 cases have currently been reported in the oral cavity2,4–19 and we are reporting the first case in the palate.\n\n\nCase report\n\nA 59-year-old female patient presented to the outpatient clinic in the Oral and Maxillofacial Surgery Department, Cairo University in January 2019 complaining of a large painless swelling in the palate (Figure 1). The patient reported that the swelling had been present in her oral cavity for 22 years. The patient’s medical and familial histories were unremarkable. Upon clinical examination the day of admission, a large palatal swelling (3 cm × 3 cm) was evident on the right side of the hard palate. The swelling was covered by normal mucosa and showed a slight bluish tinge. The lesion was not visible on radiographs.\n\nAn incisional biopsy of the lesion was performed. Hematoxylin and eosin stained sections revealed meningothelial cells arranged in lobules. The cells exhibited round to oval nuclei (Figure 2). Psammoma bodies were also present (Figure 3). No mitotic activity and no cellular atypia were found. Immunohistochemical staining for tumor-associated markers was performed to confirm a diagnosis of meningioma diagnosis and to exclude other mimic tumors as metastiatic carcinomas, schwannoma, neurofibroma, paraganglioma and perineurioma. Cells were positively stained using primary antibodies for epithelial membrane antigen (EMA) and vimentin (Figure 4a, b), but were not stained when using primary antibodies for S100, pancytokeratin, p63, chromogranin and renal cell carcinoma glycoprotein (Figure 5a–e).\n\nIndistinct cell membranes with uniform nuclei and no mitotic figures (inset; magnification, ×200).\n\nPsammoma bodies seen between meningiothelial cells (inset), (×200).\n\nMeningioma tumor cells showing a positive cytoplasmic immunohistochemical reaction for (a) epithelial membrane antigen and (b) Vimentin (magnification, ×200).\n\nMeningioma tumor cells react negatively following immunohistochemical staining for (a) renal cell carcinoma glycoprotein, (b) S100, (c) chromoginin, (d) p63, (e) PanCK (magnification, ×100).\n\nNo therapy was administered to the patient during her admission. Unfortunately, the patient did not show up for surgical excision and follow-up.\n\nM, male; F, female; RL, radiolucent; RO, radioopaque; UL, unilocular; ML, multilocular; NA, not available; NS, not stated.\n\n\nDiscussion\n\nPrimary extra-cranial meningioma is an unusual tumor, especially in the oral cavity4. The first intraoral meningioma reported was by Brown et al. in 1976, which presented as a periapical radiolucency anterior maxillary region5.\n\nTo the extent of our knowledge, 18 cases of primary meningioma in the oral cavity have been reported. Of these, 13 were in female patients, which is also true of the present case. However, the age range was large in the reported cases – between 10 and 77 years old2,4–19; in the present case, the patient was 59 years old. Regarding the reported cases of intraoral primary meningioma, 6 of the 18 were in the maxilla2,4–6,8,10,15, 10 were in the mandible7,9–14,18 and 2 in the buccal mucosa16,17. To our knowledge, we report the first case in the palate.\n\nThe histopathological criteria of extracranial meningiomas are similar to those of their intracranial counterparts. All documented cases shared the same characteristics: whorls of spindle cells or epithelioid cell proliferation and psammoma bodies. In our case, diagnosis was challenging because of the tumor’s similarity with other tumor entities of peripheral nerve origin, as well as the uncommon location of the tumor. An immunohistochemical panel of tumor-associated markers were used to confirm the diagnosis and to avoid unnecessary aggressive treatment. Most of the 18 cases reported in the literature achieved their diagnosis using immunohistochemical markers. All reported cases that used immunohistochemistry techniques to diagnose meningioma4,8–10,12,13,15,16,18,19 observed that the tumor cells stained positive for monoclonal antibodies against EMA and vimentin, with no immunoreactivity for S-100 protein, which was similar to our findings.\n\nUnfortunately, our patient did not show up for surgical excision and follow-up was not done because of loose of contact with the patient. However, most of the documented cases was treated successfully without recurrence by surgical excision. Some of studies, such as that by Rommel et al.19, preferred only to follow-up with the patient rather than conduct surgical intervention. However, others preferred to perform aggressive treatment, such as as segmental mandibulectomy or segmented resection7,11\n\nIn conclusion, meningioma is a rare intraoral benign neoplasm. Immunohistochemical markers are an important tool to achieve a final diagnosis, especially for the differentiation from histological mimic entities of peripheral nerve origin, such as perineurioma and neurothekeoma. Vimentin and EMA are the two important markers to confirm extra-cranial meningioma diagnosis from other lesions.\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\nEl-Naggar AK, Chan JK, Grandis JR, et al.: WHO Classification of Head and Neck Tumours. 4th edition. Lyon: IARC Press, 2017. Reference Source\n\nReddi SP, Strauss SI, Strauss JE, et al.: Anterior maxillary lesion. J Oral Maxillofac Surg. 1999; 57(10): 1234–8. PubMed Abstract | Publisher Full Text\n\nAgaimy A, Buslei R, Coras R, et al.: Comparative study of soft tissue perineurioma and meningioma using a five-marker immunohistochemical panel. Histopathology. 2014; 65(1): 60–70. PubMed Abstract | Publisher Full Text\n\nSimsek H, Komerik N: Ectopic Meningioma in the Maxillary Alveolar Ridge: Report of a Case with a Review of the Literature. IJEDS. 2012; 1(2): 98–101. Publisher Full Text\n\nBrown AM, Fordham KC, Lally ET: Meningioma presenting as an intraoral mass. Oral Surg Oral Med Oral Pathol. 1976; 41(6): 771–6. PubMed Abstract | Publisher Full Text\n\nSimpson MT, Sneddon KJ: Extracranial meningioma of the oral cavity. Br J Oral Maxillofac Surg. 1987; 25(6): 520–5. PubMed Abstract | Publisher Full Text\n\nLandini G, Kitano M: Meningioma of the mandible. Cancer. 1992; 69(12): 2917–20. PubMed Abstract | Publisher Full Text\n\nPfeifer JD, Ashley Hill D, Ramos CV, et al.: Meningioma Presenting as an Intraoral Mass in a Patient With Neurofibromatosis Type 1. Arch Pathol Lab Med. 2000; 124(6): 898–901. PubMed Abstract\n\nJones AC, Freedman PD: Primary extracranial meningioma of the mandible: A report of 2 cases and a review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001; 91(3): 338–41. PubMed Abstract | Publisher Full Text\n\nKubota Y, Yamashiro T, Kobayashi I, et al.: Primary meningioma of the mandible. Oral Onco Extra. 2005; 41(2): 18–21. Publisher Full Text\n\nMussak EN, Holodny AI, Karimi SK, et al.: Meningioma of the mandible: imaging with CT. AJNR Am J Neuroradiol. 2007; 28(6): 1157–9. PubMed Abstract | Publisher Full Text\n\nLell M, Tudor C, Aigner T, et al.: Primary intraosseous meningioma of the mandible: CT and MR imaging features. AJNR Am J Neuroradiol. 2007; 28(1): 129–31. PubMed Abstract\n\nMosqueda-Taylor A, Domínguez-Malagon H, Cano-Valdez AM, et al.: Primary extracranial meningioma of the mandible. Med Oral Patol Oral Cir Bucal. 2009; 14(4): E167–70. PubMed Abstract\n\nRushing EJ, Bouffard JP, McCall S, et al.: Primary extracranial meningiomas: an analysis of 146 cases. Head Neck Pathol. 2009; 3(2): 116–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinting L, Xiaofeng H, Zhiyong W, et al.: Extracranial meningioma in the maxillary region. J Craniofac Surg. 2013; 24(2): e142– 4. PubMed Abstract | Publisher Full Text\n\nMaeng JW, Kim Y, Seo J, et al.: Primary Extracranial Meningioma Presenting as a Cheek Mass. Clin Exp Otorhinolaryngol. 2013; 6(4): 266–268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRavindran Nair KS, Suresh Babu P, Manoj S, et al.: The meningioma that got cheeky. J Maxillofac Oral Surg. 2016; 15(Suppl 2): 279–281. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRege IC, Garcia RR, Mendonça EF: Primary Extracranial Meningioma: A Rare Location. Head Neck Pathol. 2017; 11(4): 561–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRommel N, Bissinger O, Rau A, et al.: Ectopic meningioma of the mandible in a 20-year-old woman: a case report and literature review. J Surg Case Rep. 2017; 2017(3): rjx047. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "59777",
"date": "25 Feb 2020",
"name": "Eman Abdelzaher",
"expertise": [
"Reviewer Expertise Neuropathology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is well written and presents an interesting case of ectopic meningioma. Some amendment is needed however.\nRadiological findings should be included. The authors merely mention that the lesion was not seen by radiology. Please clarify the technique used and relevant findings.\n\nThe grade of meningioma was not given\n\nThe differential diagnosis of meningioma from mimics was not sufficiently addressed. Different entities in the differential diagnosis were mentioned at different parts of the article without discussing the differentiating points. And the performed stains would not help in differentiating meningioma from perineurioma, both are positive for EMA and vimentin.\n\nGrammatical and spelling mistakes are noted here.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": [
{
"c_id": "5343",
"date": "06 Apr 2020",
"name": "Layla Hafed",
"role": "Author Response",
"response": "Thank you Dr. Eman for your valuable comments and for your time. We added that data and answers that you commented to the new version of the manuscript. And I will answer them here too as a respond to your report. 1- All the English language corrections were done.2- CT scan was done and there was no bone involvement.3- Meningioma grade I.4- The list of differential diagnosis was done and discussed."
}
]
},
{
"id": "59735",
"date": "02 Mar 2020",
"name": "Maha M. Abdelsalam",
"expertise": [
"Reviewer Expertise Oral Pathology",
"Oral medicine",
"medical and Dental Education"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCase Report: Case Report: Rare site for intraoral meningioma\n\nThe case report presents an extracranial meningioma of the hard palate. The patient’s gender is conformed to the tumor but there are (likely) no contributing or risk factors associated with its occurrence. The lesion is asymptomatic and incisional biopsy was examined routinely and certain microscopic features were suggestive of meningioma. The specimen was further stained with a panel of markers and was diagnosed as meningioma of the palate. Unfortunately the patient did not return for surgical removal.\n\nIs the background of the case’s history and progression described in sufficient detail?\nThe patient’s background can be improved if relevant findings are reported such as previous exposure to radiation as a potential risk for development of meningioma.\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?\nThis is one of the unfortunate incidents where patient is not committed to treatment.\nAlthough it seems that the patient is asymptomatic except for the size of the palatal swelling, absence of radiograph (even if non-indicative) and not mention neurofibromatosis-type2, undermine the reader’s understanding of the case.\n\nThe clinical picture is good and clear.\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?\n\nDiscussion is fine although it would’ve been beneficial to cite the other reported palatal lesions by Kishore A (2000) and Sinha (2002), for example.1,2\n\nThe immunohistochemistry test panel discussion would be more useful for the non-pathology reader if there were brief comment on the value of such tests of uncommon oral lesions with perplexing presentations.\n\nConsequently, a short list of lesions to be included in a general dentist’s record when similar cases are detected, would be useful.\n\nFew editing or English language corrections would help. For example “follow-up was not done because of loose of contact” in which “loss not loose” is meant. The statement is repeated in abstract and discussion and significantly alters the meaning of the sentence.\n\nIs the case presented with sufficient detail to be useful for other practitioners?\nExtracranial meningioma are rare and intraoral lesions are even rarer. It is of high pathological interest of both specialist and general practitioner to get updated on occurrence, presentation and diagnostic methods of such tumors.\n\nThe case and the tumor presented are useful, relevant and with corrections, the report would be more impactful.\n\nConclusions must be more powerful and instructive for the practitioner to:\nExpect the unexpected when similar lesions are encountered.\n\nSuggest an effective strategy to ensure patient’s return when the outcomes are suspicious.\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": "5342",
"date": "06 Apr 2020",
"name": "Layla Hafed",
"role": "Author Response",
"response": "Firstly, thank you doctor for your valuable comments. We added that data and answers that you commented to the new version of the manuscript. And I will answer them too here as a respond to your report. 1- There was no previous exposure to radiation as a potential risk for development of meningioma.This is one of the unfortunate incidents where patient is not committed to treatment. 2- CT scan was done and there was no bone involvement and neurofibromatosis-type 2 was excluded because the lesion is single and been present in her oral cavity for 22 year. 3- Discussion is fine although it would’ve been beneficial to cite the other reported palatal lesions. 4- The immunohistochemistry test panel discussion was added 5- Benign salivary gland neoplasm add to the list of lesions to be included in a general dentist’s record when similar cases are detected. 6- English language corrections were done. 7- Conclusions were also corrected to include the consequence of wrong diagnosed. Thanks again for your comments which add a lot to our knowledge and for your time too"
}
]
}
] | 1
|
https://f1000research.com/articles/9-95
|
https://f1000research.com/articles/9-93/v1
|
07 Feb 20
|
{
"type": "Brief Report",
"title": "Is lithium a potential treatment for the novel Wuhan (2019-nCoV) coronavirus? A scoping review",
"authors": [
"Jan K. Nowak",
"Jarosław Walkowiak",
"Jarosław Walkowiak"
],
"abstract": "The current rapid spread of the novel coronavirus (2019-nCoV) originating from Wuhan, China, calls for a rapid response from the research community. Lithium is widely used to treat bipolar disorder, but has been shown to exhibit antiviral activity. This brief review took a systematic approach to identify five in vitro studies reporting on the influence of lithium on coronaviral infections. We propose that in the case of urgent need, lithium be explored as a potential treatment or prophylaxis for the novel Wuhan coronavirus (2019-nCoV).",
"keywords": [
"coronavirus",
"Coronaviridae",
"Wuhan",
"2019-nCoV",
"lithium",
"lithium carbonate",
"lithium orotate",
"antiviral",
"apoptosis",
"glycogen synthase kinase 3-beta",
"GSK-3β"
],
"content": "Introduction\n\nThe current rapid spread of the novel coronavirus (2019-nCoV) originating from Wuhan, China, calls for a rapid response from the research community. Lithium is known to exhibit antiviral activity, but the knowledge of its potential as a possible therapy for coronoviral infections has not been summarized yet. The aim of this brief report is to draw attention to lithium as potential 2019-nCoV treatment and prophylaxis.\n\n\nMethods\n\nOn February 1st 2020 the following PubMed search was conducted with no language or time restrictions: (lithium and (coronavirus or *coronavirus or sarbecovirus or SARS or “severe acute respiratory syndrome” or MERS or “Middle East respiratory syndrome” or nobecovirus or merbecovirus or hibecovirus or embecovirus or andecovirus or buldecovirus or herdecovirus or moordecovirus or cegacovirus or igacovirus or “microhyla lentovirus” or milecovirus or alphaletovirus or tegacovirus or setracovirus or rhinacovirus or pedacovirus or “porcine epidemic diarrhea” or nyctacovirus or “nectalus velutinus” or myotacovirus or “myotis ricketti” or minunacovirus or minacovirus or luchacovirus or duvinacovirus or decacovirus or “Rhinolophus ferrumequinum” or “transmissible gastroenteritis virus” or “feline infectious peritonitis virus” or “canine coronavirus” or “murine hepatitis virus”)). The search yielded 45 articles, of which all the abstracts were charted and reviewed by two researchers.\n\n\nResults\n\nFive studies reporting on the influence of lithium on coronaviral infections were identified (Figure 1).\n\nIn Vero cells, lithium chloride was shown to be effective in suppressing infection with the porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family1. Not only PEDV entry and replication were inhibited in the presence of LiCl, but apoptosis as well. In MARC-145 cells, LiCl reduced the production of RNA and proteins specific to the porcine reproductive and respiratory syndrome virus. The authors, however, cautioned that the effect might have been dependent on LiCl presence during the early stages of infection and the increase of tumor necrosis factor-α2. In vitro studies of another porcine coronavirus causing transmissible gastroenteritis indicated that LiCl acts on both early and late stages of infection and inhibits apoptosis3. The same research group from Harbin in China reported earlier that LiCl reduced the cytopathic effect of the avian infectious bronchitis virus (also a coronavirus) in primary chicken embryo kidney cells4. In Vero cells, African green monkey kidney-derived epithelial cells, and immortalized chicken embryo fibroblasts LiCl suppressed the avian coronavirus infectious bronchitis. The antiviral activity of lithium was ascribed to a cellular effect5.\n\n\nDiscussion\n\nThe possible molecular mechanisms of reduced apoptosis include the inhibition of glycogen synthase kinase 3-beta (GSK-3β)6,7. Moreover, PEDV requires the PI3K/Akt/GSK-3α/β pathway, which can be targeted at GSK-3β by lithium8. Curiously, GSK-3β is required for template switching, a process seemingly indispensable for the production of coronaviral genomic RNA. The inhibition of GSK-3β prevents longer viral subgenomic mRNAs and the genomic RNA from being synthesized9. Their production would require GSK-3β-dependent phosphorylation of the viral nucleocapsid and subsequent recruitment of helicase DDX1.\n\nLithium carbonate is an orphan drug widely used in the treatment of bipolar disorder. Its safety, when used correctly, is excellent10. The main concern in the setting of an infectious disease unit would be the potential for interactions with other medication, possibly leading to the elevation of lithium levels and acute toxicity, mostly renal. This may be prevented by monitoring serum lithium concentrations. To our best knowledge, no interactions between lithium carbonate and ribavirin, lopinavir or ritonavir exist. In unconscious patients lithium carbonate could be given via a nasogastric tube. In case of lithium carbonate unavailability, lithium orotate could be explored, which, however, remains much less known to medical science despite being available as a dietary supplement. Overall, we propose that in the case of urgent need lithium be explored by physicians as a potential treatment or prophylaxis for the novel Wuhan coronavirus (2019-nCoV).\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: PRISMA ScR checklist for ‘Is lithium a potential treatment for the novel Wuhan (2019-nCoV) coronavirus? A scoping review’. https://doi.org/10.5281/zenodo.363757411.\n\nThe adapted reporting guidelines checklist is available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nLi HJ, Gao DS, Li YT, et al.: Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro. Res Vet Sci. 2018; 118: 288–94. PubMed Abstract | Publisher Full Text\n\nCui J, Xie J, Gao M, et al.: Inhibitory effects of lithium chloride on replication of type II porcine reproductive and respiratory syndrome virus in vitro. Antivir Ther. 2015; 20(6): 565–72. PubMed Abstract | Publisher Full Text\n\nRen X, Meng F, Yin J, et al.: Action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus. PLoS One. 2011; 6(5): e18669. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi J, Yin J, Sui X, et al.: Comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection in vitro. Avian Pathol. 2009; 38(3): 215–21. PubMed Abstract | Publisher Full Text\n\nHarrison SM, Tarpey I, Rothwell L, et al.: Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture. Avian Pathol. 2007; 36(2): 109–14. PubMed Abstract | Publisher Full Text\n\nZaheer A, Knight S, Zaheer A, et al.: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3. Brain Res. 2008; 1190: 206–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChoi SE, Kang Y, Jang HJ, et al.: Involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis. J Vasc Res. 2007; 44(5): 365–74. PubMed Abstract | Publisher Full Text\n\nKong N, Wu Y, Meng Q, et al.: Suppression of Virulent Porcine Epidemic Diarrhea Virus Proliferation by the PI3K/Akt/GSK-3α/β Pathway. PLoS One. 2016; 11(8): e0161508. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu CH, Chen PJ, Yeh SH: Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription. Cell Host Microbe. 2014; 16(4): 462–72. PubMed Abstract | Publisher Full Text\n\nRybakowski JK: Challenging the Negative Perception of Lithium and Optimizing Its Long-Term Administration. Front Mol Neurosci. 2018; 11: 349. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNowak JK, Walkowiak J: PRISMA Scoping Review checklist for Nowak and Walkowiak 2020. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3637575"
}
|
[
{
"id": "59741",
"date": "12 Feb 2020",
"name": "Fangqiang Wei",
"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 wide spread of infection of 2019-nCoV has arouse an international concern since its original outbreak in Wuhan, China. Scientists and health workers around the world are currently working together to wipe out the virus and the novel coronavirus pneumonia (NCP), which has killed more than a thousand lives, by far, worldwide.\n\nWith the current epidemic being so severe, it is necessary and urgent to make potentially reasonable recommendations for the treatment or prevention for 2019-nCoV or NCP. The two authors clearly proposed that lithium might be a potential treatment or prophylaxis for 2019-nCoV or NCP based on a summary of existing literature that reported the in vitro effects of lithium on coronaviral infections and discussed potential mechanisms, which sound reasonable to some extent, but still not rigorous.\n\nSpecifically, there are few related studies available and only in vitro data have been reported. The authors may need more related studies and solid evidence to support their hypothesis to make it more scientific and rigorous. As reported, lithium can be toxic due to its side effects, mainly thyroid, renal, and cognitive disturbances. Readers may wish to see more clinical information of lithium in treating viral infection cases, if not available, or in treating other diseases.\n\nIn terms of discussion, the authors reviewed some existing literature and suggested a potential mechanism of reduced apoptosis by lithium, the glycogen synthase kinase 3-beta (GSK-3β) inhibitor. The possibility that targeting at GSK-3β by lithium may potentially affect the coronavirus is an interesting topic. However, direct in vitro evidence is lacking regarding 2019-nCoV or related coronaviruses including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses.\n\nMoreover, relevant literature is still needed although the authors state that there is no interaction between lithium carbonate and ribavirin, lopinavir, or ritonavir exist. Another aspect worth noting is that the authors indicate that monitoring serum lithium concentration can be helpful in preventing side effects of lithium, however it should be emphasized that the in vivo relationship between the effective dose and toxic dose of lithium is still unclear, with some studies reporting a dose-dependent manner of the inhibitory effect of lithium in vitro. Thus, it warrants more data, both in vitro and in vivo, to clarify this issue.\n\nCollectively, this study proposes a potential role of lithium in treating or preventing 2019-nCoV or NCP with some possible mechanisms. However, by far, solid evidence is lacking to validate this hypothesis. The time of developing lithium orotate for clinical use, even in emergency, is not yet.\n\nIs the work 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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5311",
"date": "03 Apr 2020",
"name": "Jan Nowak",
"role": "Author Response",
"response": "Poznań, March 16th, 2020Dear Prof. Wei and Prof. Wang,We would like to thank you for all the comments. They helped to improve the manuscript. Please find our responses below.Sincerely yours,Jan Nowak and Jarosław Walkowiak The wide spread of infection of 2019-nCoV has arouse an international concern since its original outbreak in Wuhan, China. Scientists and health workers around the world are currently working together to wipe out the virus and the novel coronavirus pneumonia (NCP), which has killed more than a thousand lives, by far, worldwide. With the current epidemic being so severe, it is necessary and urgent to make potentially reasonable recommendations for the treatment or prevention for 2019-nCoV or NCP. The two authors clearly proposed that lithium might be a potential treatment or prophylaxis for 2019-nCoV or NCP based on a summary of existing literature that reported the in vitro effects of lithium on coronaviral infections and discussed potential mechanisms, which sound reasonable to some extent, but still not rigorous. We appreciate the critique, which has helped to improve our manuscript.Specifically, there are few related studies available and only in vitro data have been reported. The authors may need more related studies and solid evidence to support their hypothesis to make it more scientific and rigorous. More details are provided in the results. New paragraphs also discuss the antiviral activity in humans and cell cultures challenged with other viruses.As reported, lithium can be toxic due to its side effects, mainly thyroid, renal, and cognitive disturbances. Readers may wish to see more clinical information of lithium in treating viral infection cases, if not available, or in treating other diseases.Lithium cardiotoxicity is now discussed in more detail. “Lithium concentration may be, on the other hand, increased by loop or thiazide diuretics, angiotensin-converting enzyme inhibitors, and non-steroid anti-inflammatory drugs. It is also is not clear if the use of lithium would be safe in acute disease accompanied by dehydration and unstable electrolyte levels. Cardiotoxicity of lithium may occur not only with concentrations larger than 1.5 mmol/L, but also when levels of the ion rapidly change21. Although QTc prolongation is absent in most patients receiving lithium, QT dispersion ratio may increase; longer QT was also described in some cases. Concurrent use of lithium with chloroquine would need to be especially cautious in patients with QT prolongation.” A paragraph on lithium and herpes infections in patients with affective disorders was added. The results of the cited studies are the best evidence for the antiviral activity of lithium that comes from studies conducted in patients. “There is some evidence that lithium may affect the course of viral diseases in humans. In a retrospective cohort study of patients with affective disorders a decrease in the rate of recurrent labial herpes was found in the lithium group (n = 177, p < 0.001) but not in the alternative treatment group (n = 59, p = 0.53)13. In research previously conducted by Prof. J. Rybakowski at our hospital, lithium prevented labial herpes recurrence in thirteen out of 28 eligible psychiatric patients. Lithium also seemed to bring improvement in a proof-of-concept randomized double-blind placebo-controlled trial involving eleven healthy adults with recurrent HSV infections14 and in a randomized study of ten women with genital herpes conducted by the same research group from Philadelphia.” More information on the activity of lithium in other viral infections was also provided. “LiCl was shown to dose-dependently inhibit reovirus (10-60 mM)15 and food-and-mouth disease virus (10-40 mM)16. At 5 mM concentration LiCl reduced the replication of avian leukosis virus subgroup J in chicken embryo fibroblast cells17. Yet, lithium at 50 µM concentration (12-20 times smaller than usually maintained in bipolar disorder) significantly reduced hepatitis C virus copy number (P = 0.0002) in supernatant from Huh7.5 cell culture18. The latter study gives hope that lithium may indeed be efficient at clinically relevant levels.” In terms of discussion, the authors reviewed some existing literature and suggested a potential mechanism of reduced apoptosis by lithium, the glycogen synthase kinase 3-beta (GSK-3β) inhibitor. The possibility that targeting at GSK-3β by lithium may potentially affect the coronavirus is an interesting topic. However, direct in vitro evidence is lacking regarding 2019-nCoV or related coronaviruses including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses.Certainly, the evidence is lacking. The discussion is speculative. We have added the following: \"The major putative molecular mechanisms of antiviral activity and reduced apoptosis is the inhibition of glycogen synthase kinase 3-beta (GSK-3β)7,8. However, lithium also inhibits GSK-3α, inositol monophosphatases, and may indirectly act via the electrolyte balance.\" And also: “Chloroquine (hydroxychloroquine) – which is thought to be effective in COVID-201911 – was shown to inhibit GSK-3β and potentiate GSK-3β inhibition caused by lithium. This indicates that mechanistic studies could investigate not only 0.5-1.2 mM lithium, but lithium with chloroquine as well. This also brings zinc to the spotlight since zinc inhibits GSK-3β at micromolar concentrations12.” Moreover, relevant literature is still needed although the authors state that there is no interaction between lithium carbonate and ribavirin, lopinavir, or ritonavir exist. Information on tenofovir is now provided: “A randomized study in tenofovir-treated patients with HIV revealed that 24-week addition of lithium at target serum concentrations of 0.6-1.0 mmol/L was not associated with nephrotoxicity20.” Another aspect worth noting is that the authors indicate that monitoring serum lithium concentration can be helpful in preventing side effects of lithium, however it should be emphasized that the in vivo relationship between the effective dose and toxic dose of lithium is still unclear, with some studies reporting a dose-dependent manner of the inhibitory effect of lithium in vitro. Thus, it warrants more data, both in vitro and in vivo, to clarify this issue.Throughout the results section, information on the concentrations of lithium were given and commented on.Moreover, the discussion opens with: “The available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans.” Collectively, this study proposes a potential role of lithium in treating or preventing 2019-nCoV or NCP with some possible mechanisms. However, by far, solid evidence is lacking to validate this hypothesis. The time of developing lithium orotate for clinical use, even in emergency, is not yet.The reference to lithium orotate was removed.The conclusion was changed: “In the light of the reviewed data lithium appears as a possible candidate for therapy of COVID-2019. We propose mechanistic investigation of the influence of lithium (0.5-1 mM) – alone and with chloroquine or other drugs – on the SARS-CoV-2 infection.” We would like the reviewers for their input, which has guided us in improving the text. Please note that following the remarks of Prof. Beaulieu and that change of the nomenclature (2019-nCoV no longer used) we have proposed a new title, which seems more neutral and therefore more representative of the softened conclusions: “Lithium and coronaviral infections. A scoping review.” The references were updated1 Li HJ, Gao DS, Li YT, et al.: Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro. Res Vet Sci. 2018;118:288–94. 29547727 10.1016/j.rvsc.2018.03.0022 Cui J, Xie J, Gao M, et al.: Inhibitory effects of lithium chloride on replication of type II porcine reproductive and respiratory syndrome virus in vitro. Antivir Ther. 2015;20(6):565–72. 25560301 10.3851/IMP29243 Ren X, Meng F, Yin J, et al.: Action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus. PLoS One. 2011;6(5):e18669. 21573100 10.1371/journal.pone.0018669 30896054 Li J, Yin J, Sui X, et al.: Comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection in vitro. Avian Pathol. 2009;38(3):215–21. 19468938 10.1080/030794509029121845 Harrison SM, Tarpey I, Rothwell L, et al.: Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture. Avian Pathol. 2007;36(2):109–14. 17479370 10.1080/030794506011560836 Zhai X, Wang S, Zhu M, He W, Pan Z, Su S. Antiviral Effect of Lithium Chloride and Diammonium Glycyrrhizinate on Porcine Deltacoronavirus In Vitro. Pathogens. 2019 Sep;8(3):144.7Zaheer A, Knight S, Zaheer A, et al.: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3. Brain Res. 2008;1190:206–14. 18054898 10.1016/j.brainres.2007.11.011 23430018 Choi SE, Kang Y, Jang HJ, et al.: Involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis. J Vasc Res. 2007;44(5):365–74. 17483602 10.1159/0001023219 Kong N, Wu Y, Meng Q, et al.: Suppression of Virulent Porcine Epidemic Diarrhea Virus Proliferation by the PI3K/Akt/GSK-3α/β Pathway. PLoS One. 2016;11(8):e0161508. 27560518 10.1371/journal.pone.0161508 499913010 Wu CH, Chen PJ, Yeh SH: Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription. Cell Host Microbe. 2014;16(4):462–72. 25299332 10.1016/j.chom.2014.09.00911Multicenter collaboration group of Department of Science and Technology of Guangdong Province and Health Commission of Guangdong Province for chloroquine in the treatment of novel coronavirus pneumonia. [Expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia]. Zhonghua Jie He He Hu Xi Za Zhi. 2020 Mar 12;43(3):185–8.12Eldar-Finkelman H, Martinez A. GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS. Front Mol Neurosci. 2011;4:32.13Amsterdam JD, Maislin G, Rybakowski J. A possible antiviral action of lithium carbonate in herpes simplex virus infections. Biol Psychiatry. 1990 Feb 15;27(4):447–53.14Amsterdam JD, Maislin G, Hooper MB. Suppression of herpes simplex virus infections with oral lithium carbonate--a possible antiviral activity. Pharmacotherapy. 1996 Dec;16(6):1070–5.15Chen Y, Kong D, Cai G, Jiang Z, Jiao Y, Shi Y, et al. Novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro. Microb Pathog. 2016 Apr;93:152–7.16Zhao F-R, Xie Y-L, Liu Z-Z, Shao J-J, Li S-F, Zhang Y-G, et al. Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro. J Med Virol. 2017;89(11):2041–6.17Qian K, Cheng X, Zhang D, Shao H, Yao Y, Nair V, et al. Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture. Arch Virol. 2018 Apr 1;163(4):987–95.18Sarhan MA, Abdel-Hakeem MS, Mason AL, Tyrrell DL, Houghton M. Glycogen synthase kinase 3β inhibitors prevent hepatitis C virus release/assembly through perturbation of lipid metabolism. Sci Rep [Internet]. 2017 May 31 [cited 2020 Mar 14];7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451429/19 Rybakowski JK: Challenging the Negative Perception of Lithium and Optimizing Its Long-Term Administration. Front Mol Neurosci. 2018;11:349. 30333722 10.3389/fnmol.2018.00349 617599420Decloedt EH, Lesosky M, Maartens G, Joska JA. Renal safety of lithium in HIV-infected patients established on tenofovir disoproxil fumarate containing antiretroviral therapy: analysis from a randomized placebo-controlled trial. AIDS Res Ther. 2017 Feb 4;14(1):6.21Mehta N, Vannozzi R. Lithium-induced electrocardiographic changes: A complete review. Clinical Cardiology. 2017;40(12):1363–7.22 Nowak JK, Walkowiak J: PRISMA Scoping Review checklist for Nowak and Walkowiak 2020. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3637575"
}
]
},
{
"id": "59736",
"date": "21 Feb 2020",
"name": "Jean-Martin Beaulieu",
"expertise": [
"Reviewer Expertise Gsk3 signaling"
],
"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 identified five previous studies reporting an effect of lithium (mostly LiCl) in corona virus in cellular systems. This is obviously a very timely question. All studies point toward beneficial effects of lithium and thus underscore the possible beneficial effect of targeting lithium sensitive biochemical pathways, namely GSK3 mediated signaling for corona virus treatment or prophylaxis.\nAs a technical issue lithium not only targets GSK3b but also GSK3-alpha and inositol monophosphatases. So the emphasis on GSK3-beta may be a bit premature.\n\nA more important issue is that none of the studies shown an effect of lithium at a 1-1.5mM concentrations. Effects are reported at Li+ concentrations that are 5mM or higher. These concentrations are not toxic for cells in culture. However, in humans, serum lithium concentration above 1.5-2.0mM (or mEq, which stands for the mM concentration of the lithium ion) are considered toxic (Haussmann et al., 20151).\nThe prescription of lithium in the context of the current epidemic thus appears not to be supportable by the findings. The cure may kill the patients.\n\nMore detailed studies using lithium in animal models at tolerable concentrations would thus be needed.\n\nUnfortunately these limitations are not addressed in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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? No",
"responses": [
{
"c_id": "5312",
"date": "03 Apr 2020",
"name": "Jan Nowak",
"role": "Author Response",
"response": "Poznań, March 16th, 2020Dear Prof. Beaulieu,We are grateful for the comments that you have provided. They helped to improve our manuscript. Please find our responses below.Sincerely yours,Jan Nowak and Jarosław Walkowiak The authors identified five previous studies reporting an effect of lithium (mostly LiCl) in corona virus in cellular systems. This is obviously a very timely question. All studies point toward beneficial effects of lithium and thus underscore the possible beneficial effect of targeting lithium sensitive biochemical pathways, namely GSK3 mediated signaling for corona virus treatment or prophylaxis.As a technical issue lithium not only targets GSK3b but also GSK3-alpha and inositol monophosphatases. So the emphasis on GSK3-beta may be a bit premature. We thank the reviewer for this comment. This is now referred to early in the discussion: “The major putative molecular mechanisms of antiviral activity and reduced apoptosis is the inhibition of glycogen synthase kinase 3-beta (GSK-3β)7,8. However, lithium also inhibits GSK-3α, inositol monophosphatases, and may indirectly act via the electrolyte balance.” A more important issue is that none of the studies shown an effect of lithium at a 1-1.5mM concentrations. Effects are reported at Li+ concentrations that are 5mM or higher. These concentrations are not toxic for cells in culture. However, in humans, serum lithium concentration above 1.5-2.0mM (or mEq, which stands for the mM concentration of the lithium ion) are considered toxic (Haussmann et al., 20151).This important comment has triggered a number of major changes in the manuscript. In order not to mislead the readers we propose a new title, which is more neutral: “Lithium and coronaviral infections. A scoping review.” The conclusion of the abstract was changed: “We propose mechanistic investigation of the influence of lithium – alone and with chloroquine – on the SARS-CoV-2 infection.” The conclusion at the end of the discuss was altered: “In the light of the reviewed data lithium appears as a possible candidate for therapy of COVID-2019. We propose mechanistic investigation of the influence of lithium (0.5-1 mM) – alone and with chloroquine or other drugs – on the SARS-CoV-2 infection.” Throughout the results section we now report and comment on LiCl concentrations, which are clearly above the levels, which are safe for humans. Examples: “In Vero cells, lithium chloride (investigated at 1–15 mM) was shown to be dose-dependently effective in suppressing infection with the porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family1.“ “Yet, LiCl at 1 mM (safe in patients) was not effective. At 5 mM LiCl reduced viral RNA levels by 30% (p < 0.001).” “The relative viral mRNA level decreased by more than 30% (p < 0.001) at the concentration of 10 mM and by 50% at 20 mM (p < 0.001).” “Both virus titer reduction and cell survival at 70–90% were achieved with LiCl at 25 mM (10–50% at 5 mM).” “The results suggest that the dose of 5 mM was beneficial (20% inhibition) when applied one hour after infection, but not 8 hours post infection.” “Relative virus titers in both cell lines were reduced by at least 45% at 5 mM and 70–90% at 10 mM. Viral mRNA concentration decreased 20 times in both cell types cultured with 5 mM LiCl.” “One study was identified outside the main search reports on the activity of high LiCl concentrations (10-60 mM) against porcine deltacoronavirus: at 10 mM 50% relative mRNA reduction was found with no accompanying effect on the viral titer6.” Crucially, the discussion now opens with: “The available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans.” The prescription of lithium in the context of the current epidemic thus appears not to be supportable by the findings. The cure may kill the patients. As cited above, the discussion now starts by stating: “The available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans.” However, hypothesizing that lithium could be useful in treating viral infections is now supported by some other evidence: “There is some evidence that lithium may affect the course of viral diseases in humans. In a retrospective cohort study of patients with affective disorders a decrease in the rate of recurrent labial herpes was found in the lithium group (n = 177, p < 0.001) but not in the alternative treatment group (n = 59, p = 0.53)13. In research previously conducted by Prof. J. Rybakowski at our hospital, lithium prevented labial herpes recurrence in thirteen out of 28 eligible psychiatric patients. Lithium also seemed to bring improvement in a proof-of-concept randomized double-blind placebo-controlled trial involving eleven healthy adults with recurrent HSV infections14 and in a randomized study of ten women with genital herpes conducted by the same research group from Philadelphia.” Therefore it seems that in some instances lithium exhibits antiviral activity at concentrations, which are safe and maintained long-term (for years) in patients with affective disorders.Additionally, the discussion of lithium safety is now broader. “Lithium concentration may be, on the other hand, increased by loop or thiazide diuretics, angiotensin-converting enzyme inhibitors, and non-steroid anti-inflammatory drugs. It is also is not clear if the use of lithium would be safe in acute disease accompanied by dehydration and unstable electrolyte levels. Cardiotoxicity of lithium may occur not only with concentrations larger than 1.5 mmol/L, but also when levels of the ion rapidly change21. Although QTc prolongation is absent in most patients receiving lithium, QT dispersion ratio may increase; longer QT was also described in some cases. Concurrent use of lithium with chloroquine would need to be especially cautious in patients with QT prolongation.” More detailed studies using lithium in animal models at tolerable concentrations would thus be needed. As we mentioned, this has been adapted to be the conclusion of our study. “In the light of the reviewed data lithium appears as a possible candidate for therapy of COVID-2019. We propose mechanistic investigation of the influence of lithium (0.5-1 mM) – alone and with chloroquine or other drugs – on the SARS-CoV-2 infection.” Unfortunately these limitations are not addressed in the manuscript.We thank for the critique, which has helped to transform our manuscript. The references were updated1 Li HJ, Gao DS, Li YT, et al.: Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro. Res Vet Sci. 2018;118:288–94. 29547727 10.1016/j.rvsc.2018.03.0022 Cui J, Xie J, Gao M, et al.: Inhibitory effects of lithium chloride on replication of type II porcine reproductive and respiratory syndrome virus in vitro. Antivir Ther. 2015;20(6):565–72. 25560301 10.3851/IMP29243 Ren X, Meng F, Yin J, et al.: Action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus. PLoS One. 2011;6(5):e18669. 21573100 10.1371/journal.pone.0018669 30896054 Li J, Yin J, Sui X, et al.: Comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection in vitro. Avian Pathol. 2009;38(3):215–21. 19468938 10.1080/030794509029121845 Harrison SM, Tarpey I, Rothwell L, et al.: Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture. Avian Pathol. 2007;36(2):109–14. 17479370 10.1080/030794506011560836 Zhai X, Wang S, Zhu M, He W, Pan Z, Su S. Antiviral Effect of Lithium Chloride and Diammonium Glycyrrhizinate on Porcine Deltacoronavirus In Vitro. Pathogens. 2019 Sep;8(3):144.7Zaheer A, Knight S, Zaheer A, et al.: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3. Brain Res. 2008;1190:206–14. 18054898 10.1016/j.brainres.2007.11.011 23430018 Choi SE, Kang Y, Jang HJ, et al.: Involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis. J Vasc Res. 2007;44(5):365–74. 17483602 10.1159/0001023219 Kong N, Wu Y, Meng Q, et al.: Suppression of Virulent Porcine Epidemic Diarrhea Virus Proliferation by the PI3K/Akt/GSK-3α/β Pathway. PLoS One. 2016;11(8):e0161508. 27560518 10.1371/journal.pone.0161508 499913010 Wu CH, Chen PJ, Yeh SH: Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription. Cell Host Microbe. 2014;16(4):462–72. 25299332 10.1016/j.chom.2014.09.00911Multicenter collaboration group of Department of Science and Technology of Guangdong Province and Health Commission of Guangdong Province for chloroquine in the treatment of novel coronavirus pneumonia. [Expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia]. Zhonghua Jie He He Hu Xi Za Zhi. 2020 Mar 12;43(3):185–8.12Eldar-Finkelman H, Martinez A. GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS. Front Mol Neurosci. 2011;4:32.13Amsterdam JD, Maislin G, Rybakowski J. A possible antiviral action of lithium carbonate in herpes simplex virus infections. Biol Psychiatry. 1990 Feb 15;27(4):447–53.14Amsterdam JD, Maislin G, Hooper MB. Suppression of herpes simplex virus infections with oral lithium carbonate--a possible antiviral activity. Pharmacotherapy. 1996 Dec;16(6):1070–5.15Chen Y, Kong D, Cai G, Jiang Z, Jiao Y, Shi Y, et al. Novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro. Microb Pathog. 2016 Apr;93:152–7.16Zhao F-R, Xie Y-L, Liu Z-Z, Shao J-J, Li S-F, Zhang Y-G, et al. Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro. J Med Virol. 2017;89(11):2041–6.17Qian K, Cheng X, Zhang D, Shao H, Yao Y, Nair V, et al. Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture. Arch Virol. 2018 Apr 1;163(4):987–95.18Sarhan MA, Abdel-Hakeem MS, Mason AL, Tyrrell DL, Houghton M. Glycogen synthase kinase 3β inhibitors prevent hepatitis C virus release/assembly through perturbation of lipid metabolism. Sci Rep [Internet]. 2017 May 31 [cited 2020 Mar 14];7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451429/19 Rybakowski JK: Challenging the Negative Perception of Lithium and Optimizing Its Long-Term Administration. Front Mol Neurosci. 2018;11:349. 30333722 10.3389/fnmol.2018.00349 617599420Decloedt EH, Lesosky M, Maartens G, Joska JA. Renal safety of lithium in HIV-infected patients established on tenofovir disoproxil fumarate containing antiretroviral therapy: analysis from a randomized placebo-controlled trial. AIDS Res Ther. 2017 Feb 4;14(1):6.21Mehta N, Vannozzi R. Lithium-induced electrocardiographic changes: A complete review. Clinical Cardiology. 2017;40(12):1363–7.22 Nowak JK, Walkowiak J: PRISMA Scoping Review checklist for Nowak and Walkowiak 2020. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3637575"
}
]
}
] | 1
|
https://f1000research.com/articles/9-93
|
https://f1000research.com/articles/9-232/v1
|
02 Apr 20
|
{
"type": "Research Article",
"title": "Prediction of local COVID-19 spread in Heidelberg",
"authors": [
"Lisa Koeppel",
"Claudius Gottschalk",
"Andreas Welker",
"Britta Knorr",
"Claudia M. Denkinger",
"Claudius Gottschalk",
"Andreas Welker",
"Britta Knorr",
"Claudia M. Denkinger"
],
"abstract": "Since the first identified case of COVID-19 in Wuhan, China, the disease has developed into a pandemic, imposing a major challenge for health authorities and hospitals worldwide. Mathematical transmission models can help hospitals to anticipate and prepare for an upcoming wave of patients by forecasting the time and severity of infections. Taking the city of Heidelberg as an example, we predict the ongoing spread of the disease for the next months including hospital and ventilator capacity and consider the possible impact of currently imposed countermeasures.",
"keywords": [
"COVID-19",
"compartmental model",
"mortality",
"hospitalization"
],
"content": "Introduction\n\nSince the first appearance of the novel coronavirus disease (COVID-19) in Wuhan, China, in December 2019, the disease has spread worldwide to be classified as a pandemic by the WHO on 11 March 2020.\n\nIn Germany the first confirmed COVID-19 case was recorded on 28 January 2020. Contact tracing and isolating of affected individuals interrupted the chains of transmission. However, with tourists returning from holidays in high-risk regions of northern Italy combined with a carnival celebration near Cologne, the virus spread to 13 out of 16 federal states within only a month1. The exponential increase in new confirmed cases in Germany has reached 16,290 positively tested cases by 20 March 2020, with many more cases presumed undetected due to a possible transmission from minimally symptomatic or asymptomatic persons carrying the virus2,3.\n\nThe spread of COVID-19 in other countries, such as China and Italy, indicate the major challenges German hospitals, especially intensive care facilities, will have to face in the weeks to come. On 17 March 2020, the Robert Koch Institute declared the population risk as “high”. For the elderly and individuals with pre-existing health conditions, there is an increased probability of severe disease requiring hospitalization and critical care treatment, as well as higher mortality rates4.\n\nThe spread of the disease varies locally and depends on several factors, such as the temporal progression of the infection in the specified region, the size and the age distribution of the population, the available capacities and the implemented countermeasures. Thus, to prepare hospitals and assist health authorities in the most efficient way, the resulting challenge and burden on the health care system should be modelled locally. As an exemplary case, in this manuscript we are concerned with the spread of the COVID-19 epidemic for the city of Heidelberg in Germany.\n\nThe first COVID-19 positive case in Heidelberg was confirmed on 28 February 2020, with eight more cases sporadically appearing in the following two weeks5. The vast majority were home-comers from vacations in a high-risk region in Italy and their immediate contacts. All positive tested individuals were isolated and several hundred contacts were traced and quarantined to prevent further spread. Only thereafter, the total numbers of positively tested COVID-19 cases rose to 20 on March 15, and 34 on March 16.\n\nThe capabilities to cope with the increasing burden on the health system on a local scale depend on the preparedness of available local resources in the hospitals and the implemented countermeasures (isolation, quarantine, social distancing) to slow down the disease spread. In such an extraordinary situation, it is crucial for hospitals to rely on future predictions about the upcoming need of beds as well as ventilators. Predictions from mathematical modelling can assist authorities in their decision making on suitable countermeasures to slow down the viral transmission and prepare the health systems.\n\n\nMethods\n\nWe model the transmission dynamics of the COVID-19 outbreak by an individual level susceptible (S) – exposed (E) – infected (I) – removed (R; SEIR) compartmental model. The transitions between the compartments are described through the following set of differential equations\n\n\n\n\n\n\n\n\n\nWhere γ is the inverse of the infectious period and κ is the inverse of the incubation period. Extended data, Table 16 lists all parameter choices for our model. For the main part of this manuscript we assume an incubation period of 5 days7 and an infectious period each of 2.9 days8. The infectiousness of the disease is given by R0 and is defined as the mean number of people that get infected by a single infected individual over the whole infectious period. Due to the limited data about the COVID-19 pandemic, there is much uncertainty in the estimation of R0. As baseline we follow the value of 2.2 obtained from the COVID-19 outbreak on the Diamond Princess ship9. For a sensitivity analysis, we also consider a lower R0 of 1.5 with respect to the estimation of Du et al. (2020)10 and a more severe outbreak progression with 3 in accordance to the estimation of R0 ranging between 2.2 and 3.6 for the SARS epidemic in 200311.\n\nAccording to the office for statistics and urban development Heidelberg12, for our model we assume a closed population of 150,000, without individual entering or leaving the system. We consider this as a realistic scenario because we do not expect major demographic changes to occur within the six months of the projections.\n\nThe true number of initial infected individuals remains unknown due to the asymptomatic progression in some cases. Furthermore, due to limited testing capacities, only high-risk individuals (i.e. people who had prior close contact to infected individuals or people returning from a high risk region according to the WHO definition) were tested, resulting in missing positive cases in the population that resulted from possible community transmission. To capture this uncertainty, we assume that the outbreak starts on 12 March 2020 with 10 initial infectious individuals who have not been isolated and are able to transmit the disease.\n\n\nResults\n\nFor now, we assume that no countermeasures have been set in place and predict the future dynamics of the outbreak for the upcoming period of 6 months. Figure 1 displays the number of infected individuals per day for varying values of R0. In the baseline scenario (R0 = 2.2) the peak of the infection will be 10196 individuals infected on 26 May and a total outbreak size of 126,574 (84%) of the population infected after six months. This aligns with estimates for the proportion of British people get infected if no countermeasures are put in place13.\n\nIn all simulations, the number of initial infections was 10 and the mean incubation and infectious period is 5 days.\n\nIn the mild outbreak scenario, there will be 77952 (52%) total infections by 12 September 2020, whereas in the severe scenario we will have a rapid growth and a shorter doubling of infections time with 141,084 (94%) infections in total.\n\nFor sensitivity analysis, we also investigated, with respect to expert opinion, shortening and prolonging the incubation period [3.5–5.5 days] as well as the infectious period [1.5–4.5 days] (see Extended data, Figure A16). The model showed more robustness to this kind of variation on the overall outbreak curve with a shift in max 42 days for the peak of the outbreak curve and difference of 8856 infections during peak time (compared to 90 days and 12,635 when varying R0).\n\nWhen varying the number of first infected individuals, the dynamics of the outbreak stay the same. The more infected individuals we start the simulation with, the further we assume the outbreak has progressed already (see Extended data, Figure A26).\n\nFrom these scenarios, it is obvious that lowering R0 leads to a lowering of the final size of the epidemic, prolonging the peaking and minimizing the peak. The latter is the most important, as it avoids an outbreak trajectory, in which a large number of people get sick at the same time with overwhelming effects for the health system.\n\nIn the hope to lower the amount of secondary cases resulting from one primary case, on 22 March 2020, the German government announced the implementation of extraordinary measures to reduce social contact and thus transmission for at least two weeks. These include regulations on social distancing such as the closure of schools, kindergartens, shops, restaurants, imposing a visiting ban for hospitals and elderly homes, as well as preventing transmission amplification events and social interactions in public with more than two persons. Such countermeasures can be considered in the model by diminishing the transmission intensity R0.\n\nFigure 2 displays the progression of the outbreak in Heidelberg with respect to different reductions in social contact and thus disease transmission from Monday, March 23 onwards. It illustrates, that restricting social contact will have a major impact on the number of infected individuals per day, on the overall outbreak size and flatten the curve. In fact, a permanent transmission reduction by 30% will yield a total of 89,022 infected individuals by mid-September and 3,877 during the peak time (Figure 2a). Decreasing social contact by 50% will massively flatten the curve, with a total of 6,927 and 223 infected individuals at peak time. This will not stop the outbreak, but it will slow it down to a major extent. Considering the transmission on a scale of one year in time, with the permanent reduction of 50% of transmission, we will have 15% of the population of Heidelberg infected.\n\nThe baseline transmission rate is R0 = 2.2. (a) Permanent reduction of disease transmission over the next six months. (b) After three weeks of reduced transmission, transmission increases again by 50% of the reduction.\n\nHowever, such a permanent lock-down of the population will not be feasible due to economical and psychological health reasons. Thus, in Figure 2b, we assume that after three weeks social life will be partially resumed and transmission rates will increase again by 50%. In this case, a transmission reduction by 70% at first with a following increase in contact rates (transmission reduction lowered to 35%) will lead to a total of 48,468 people infected by September. Compared to the worst-case scenario (permanent R0 = 2.2) this is a decrease of 62% (78,106) in the total number of people infected.\n\nIf after three weeks contact rates will revert to their original state, the outbreak curve will only be shifted by weeks with no effect on the height nor on the outbreak size (see Extended data, Figure A36).\n\nAs the severity of the disease and thus the number of expected deaths from the COVID-19 outbreak depends on the age of the individual, we obtained the specific age distribution for the city of Heidelberg: <55 (72.4%), 55–65 (11.06%), 65–75 (7.77%), and >75 years of age (8.77%)12. Adapting from Dowd et al. (2020)14, the mortality probabilities for each age category are 0.1% (<55), 1.5% (55–65), 7% (65–75), and 20% (>75 years of age). By means of these percentages we calculated the cumulative number of deaths in Heidelberg as proportions of the people in the “Removed (R)” compartment over time (Figure 3) for different scenarios of transmission reduction. We model the delay between end of infectious period and time of death by 6.2 days and follow Sanche et al. (2020)15, who assume that the duration from hospital admittance to death is 11.2 days.\n\nAs preparation for the health system to plan the bed and ventilator capacity that needs to be enabled, it is important to predict the number of patients who require hospitalization and ventilation.\n\nIt is difficult to give estimates for the proportion of hospitalization from all infected individuals, because there is an inherent bias in the existing data16. In the beginning of an epidemic, people are hospitalized for isolation purposes but not because of their severe symptoms. Besides, due to asymptomatic or minimally symptomatic infections that do not present for testing, the true number of infections remains unknown and thus estimates of hospitalization might be overly pessimistic. For this simulation study, we nevertheless assume, that 5% of all infected individuals need hospitalization. Because the duration of hospitalization varies in the literature, we take the smaller and more conservative estimate of 11.5 days15,17,18 (Figure 4a). As the curves are proportions of the number of infected individuals, changing the proportion of hospitalizations will only change the scale of the plot. A sensitivity analysis reducing the duration of hospitalization to seven days is shown in Figure A3 in the appendix.\n\nFor calculating the number of ventilators needed, we assume that 90% of all people who died from the disease were ventilated before. The remaining 10% possess a patient do not resuscitate/do not intubate (DNR/DNI) status. We further assume that 60% of ventilated die. This leads to 1.5 (0.9/0.6) people ventilated per death count. According to Chen et al. (2020)19, the average duration of ventilation is 3-20 days. Supposing a conservative estimate of 7 days, we can model the capacity in the Intensive Care Unit given the calculated death numbers.\n\nAccording to the Science Media Center Germany, routine capacity for ventilation of the University Hospital Heidelberg is 112 spaces20. Even if we unrealistically assume that all spaces can be used for COVID-19 patients, the capacity will not be enough within the following six months, unless we have a permanent reduction in the disease transmission by 50% (Figure 4b).\n\n\nDiscussion\n\nThis manuscript modelled the future of COVID-19 spread within the city of Heidelberg and showed the various possible scenarios over the coming weeks and months. Considering the current exponential growth of cases in Germany1, we have to assume a current R0 between 2.2. and 3.\n\nOur model has several limitations. Our model does not account for the percentage of people with previous conditions who are at increased risk. We also did not include any movement data in our model and did not account for tourists visiting Heidelberg, which we assume to be a realistic scenario given that governmental restrictions will keep movement to a minimum in the following months. We also did not account for deaths due to selective treatment of COVID-19 patients over others with different health conditions when bed capacity is exceeded.\n\nAlthough the number of infected individuals and therefore the number of deaths can be reduced by prolonging the outbreak, severe impacts on the economy as well as effects on psychological and social life can be expected if restrictions on social behaviour and interactions will be imposed for a longer time. In an optimistic scenario with a permanent reduction in transmission, in twelve months 50% of the population in Heidelberg will be infected. This clearly states the urgency for developing and distributing vaccines among the population.\n\nThe predictions of our model are estimations and include inherent uncertainty because the model parameters are derived from only limited clinical and epidemiological data of the COVID-19 outbreak dynamics and estimates thereof. Moreover, the future behaviour of the population, their adherence to movement and social contact restriction and the timely development of a suitable vaccine determine the ongoing of the spread. To better understand, where we are on the epidemic curve, requires more testing. The currently implemented restrictive testing strategy focuses on people returning from high-risk regions and their immediate contacts and does not capture ongoing community transmission. Community transmission is likely going to be more relevant for transmission at this stage of the epidemic as it is more difficult capture and more dispersed. Currently available testing capacity would not allow testing of all persons with respiratory symptoms in the city of Heidelberg, but at a minimum sentinel testing of a representative subset of the population should be considered. Ultimately a seroprevalence survey will be necessary to completely understand the true number of cases in the population.\n\nWe anticipate the modelling outcomes can be useful for health authorities in their decision-making process to plan resources and bed capacities ahead of time and implement suitable preparations to meet the upcoming medical need.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Prediction of local COVID-19 spread in Heidelberg - Supplementary Material. https://doi.org/10.6084/m9.figshare.120386646.\n\nThis project contains the following extended data:\n\nFigure A1: 500 Simulations with fixed R0=2.2 and changing values for κ~U [0.1818,0.2857] and γ~U [0.2222,0.6666].\n\nFigure A2: Outbreak simulations with varying number of first infected individuals on 12 March 2020.\n\nFigure A3: Outbreak simulations with different reductions in transmission from Monday, 23 March 2020.\n\nTable 1: Choices of model parameters.\n\nExtended are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nRobert Koch-Institute: COVID-19: Fallzahlen in Deutschland und weltweit. Robert Koch-Institute. 2020; (accessed March 23, 2020). Reference Source\n\nNishiura H, Kobayashi T, Miyama T, et al.: Estimation of the asymptomatic ratio of novel coronavirus infections (COVID-19). medRxiv. 2020. Publisher Full Text\n\nJohn Hopkins University (JHU): Coronavirus COVID-19 Global Cases by the Center for Systems Science and Engineering (CSSE). (accessed March 22, 2020). Reference Source\n\nRobert Koch-Institute: Risikobewertung zu COVID-19. Robert Koch-Institute, 2020; (accessed March 17, 2020). Reference Source\n\nPersonal communication with Andreas Welker.\n\nKoeppel L, Gottschalk C, Welker A, et al.: Prediction of local COVID-19 spread in Heidelberg - Supplementary Material. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12038664.v2\n\nWorld Health Organization: Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19). World Health Organization, 2020; (accessed March 17, 2020). Reference Source\n\nLiu T, Hu J, Kang M, et al.: Transmission dynamics of 2019 novel coronavirus (2019-nCoV). 2020. Publisher Full Text\n\nLi Q, Guan X, Wu P, et al.: Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N Engl J Med. 2020; 382(13): 1199–1207. PubMed Abstract | Publisher Full Text\n\nDu Z, Xu X, Wu Y, et al.: Serial Interval of COVID-19 among Publicly Reported Confirmed Cases. Emerg Infect Dis. 2020; 26(6). PubMed Abstract | Publisher Full Text\n\nLipsitch M, Cohen T, Cooper B, et al.: Transmission dynamics and control of severe acute respiratory syndrome. Science. 2003; 300(5627): 1966–1970. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmt fürStadtentwicklung und Statistik, Heidelberg. Eigene Fortschreibung. 2020. Reference Source\n\nFerguson NM, Laydon D, Nedjati-Gilani G, et al.: Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand. London: Imperial College COVID-19 Response Team, March,16. 2020. Reference Source\n\nDowd JB, Rotondi V, Adriano L, et al.: Demographic science aids in understanding the spread and fatality rates of COVID-19. medRxiv. 2020. 03.15.20036293. Publisher Full Text\n\nSanche S, Lin YT, Xu C, et al.: The novel coronavirus, 2019-nCoV, is highly contagious and more infectious than initially estimated. arXiv preprint arXiv: 2002.03268. 2020. Reference Source\n\nRobert Koch-Institute: Anteil der Hospitalisierten unter den Erkrankten. Robert Koch-Institute. 2020. (accessed March 22, 2020). Reference Source\n\nCai Q, Huang D, Ou P, et al.: COVID-19 in a Designated Infectious Diseases Hospital Outside Hubei Province, China. MedRxiv. 2020. Publisher Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med. 2020. PubMed Abstract | Publisher Full Text\n\nChen N, Zhou M, Dong X, et al.: Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020; 395(10223): 507–513. PubMed Abstract | Publisher Full Text\n\nScience Media Center Germany: Vorbereitungen in Krankenhäusern auf COVID-19. Science Media Center Germany. 2020. Reference Source"
}
|
[
{
"id": "68118",
"date": "10 Aug 2020",
"name": "Mohammad Reza Mahmoud",
"expertise": [
"Reviewer Expertise Data Analysis",
"Statistics",
"Data Modelling"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this work, the authors predict the ongoing spread of the disease including hospital and ventilator capacity for the next months in the city of Heidelberg and consider the possible impact of currently imposed countermeasures.\nThis is an interesting contribution to the existing literature, but the paper suffers from several shortcomings listed in the following comments:\nThe paper should be checked by a native.\n\nA Conclusion section should be added.\n\nThe novelty and contribution should be clearly bolded.\n\nThe introduction should be updated by recent researches. The authors should consider some works about statistical methods (including time series models or regression models) that can be applied to model and predict medical datasets.\n\nComparative Analysis should be given in more details.\n\nIt’s better to suggest some subjects for future works.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "68119",
"date": "10 Aug 2020",
"name": "Arindam Basu",
"expertise": [
"Reviewer Expertise Epidemiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors used a compartmental model that they referred to as a SEIR model where they divided the population into four compartments: Susceptible (S), Exposed (E), Infected (I), and Removed (R; Removed == \"Recovered\" or \"Died\", hence cannot be reinfected, although this assumption could be modified but for the sake of argument, we will accept that those who recovered could not be reinfected and hence removed).\n\nNow the model could be specified differently as their scheme of the equations was not clear to me. The susceptibles pool depleted at a constant rate of exposure (typically mixing rates and rate of contact with another infected person), but they seem to have substituted and mixed up with an SIR model (susceptible, infected, and Recovered/Removed model).\n\nS -> E -> I -> R should be the correct order of their model specification, so one way to formulate would be dS/dt = - Rate of Exposed dE/dt = Rate of Exposed - Rate of Infected dI/dt = Rate of Infected - Rate of removal dR/dt = Rate of accumulation of removed persons from the pool\nBut from reading their formulae it does not appear that they went with these equations, nor did they explain the basis or provide a reproducible code.\nHence, although the epidemic curves look plausible, it is difficult to interpret how they were specified or their true significance. The authors may want to clarify this aspect of the methods 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "69089",
"date": "24 Aug 2020",
"name": "Farhad Hatami",
"expertise": [
"Reviewer Expertise Biostatistics",
"Machine Learning"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn Koppel et al., the authors propose a SEIR (Susceptible, Exposed, Infected, Removed or Recovered) differential equations model estimating the spread of COVID-19 in the city of Heidelberg.\nThe proposed modelling approach is performed on a closed population with no new entry/leave. As declared by the authors, a more realistic approach could extend the current model with taking into account the scenario where people with different underlying health conditions/immune systems would have an unexpectedly higher rate of susceptibility/infection, or a second scenario where a \"recovered\" case does not necessarily mean \"not susceptible\" anymore. However, for the sake of this paper, the deployed model benefits from high robustness which makes it a reliable prediction algorithm.\nA sensitivity analysis of the model has been performed via applying on a range of assumptions for countermeasures and infection rates. As a result, changing the number of initially infected individuals at the beginning of the pandemic seems to only shift the distribution linearly with no effect on the peak. Considering the limitations in the intensive care unit, a set of policies have been introduced to help the authorities in Germany.\n\nWhile using a well-known robust algorithm, and bearing in mind that this is not supposed to be a methodological research, the paper still lacks some validation tests. A validation test on the existing benchmarking experiment can be carried out in several ways. At the time of this review, there might be access to the real data of COVID-19 spread in Heidelberg in order to compare with the estimated results. Alternatively, the same approach (of course with a different rate of daily infection) could be applied on a data of SARS to test the performance of the model.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-232
|
https://f1000research.com/articles/9-229/v1
|
02 Apr 20
|
{
"type": "Review",
"title": "Oral squamous cell carcinoma: metastasis, potentially associated malignant disorders, etiology and recent advancements in diagnosis",
"authors": [
"Amr Bugshan",
"Imran Farooq",
"Amr Bugshan"
],
"abstract": "Oral squamous cell carcinoma (OSCC) is a commonly occurring head and neck cancer. It has a high prevalence in certain parts of the world, and is associated with a high mortality rate. In this review, we describe metastasis related to OSCC, and disorders that could lead to OSCC with common etiological factors. In addition, a brief account of the diagnosis of OSCC and role of salivary biomarkers in its early detection has also been highlighted. Google Scholar and PubMed search engines were searched with keywords including “oral squamous cell carcinoma”, “OSCC”, “oral cancer”, “potentially malignant disorders in oral cavity”, “etiological factors of OSCC”, “diagnosis of OSCC”, and “salivary biomarkers and OSCC” to gather the literature for this review. The review concludes that OSCC has the potential for regional as well as distant metastasis, and many potentially malignant diseases can transform into OSCC with the help of various etiological factors. Diagnosis of OSCC involves traditional biopsy, but salivary biomarkers could also be utilized for early recognition.",
"keywords": [
"Oral squamous cell carcinoma",
"Metastasis",
"Potentially malignant disorders",
"Etiological factors of OSCC",
"Diagnosis of OSCC"
],
"content": "Introduction\n\nOne of the commonest forms of cancer is head and neck cancer1. Its prevalence is different in various parts of the world; in unindustrialized countries, like India, it is the cancer most commonly diagnosed in male patients whereas in the Western world, it is responsible for 1–4% of all cancers2. Lip, oral cavity, and oropharynx combined were responsible for about 4,47,751 new cancer cases with an estimated 2,28,389 deaths in 2018, which accounts for 2.4% of all cancer deaths3. Among other cancers, head and neck cancer is fourteenth in terms of incidence but thirteenth in terms of mortality3. The Asian continent has the highest incidence and mortality rates of oral cavity and oropharynx cancers among all other countries4. More than 90% of cancer cases in head and neck region are OSCCs (Figure 1)5. OSCC develops in the oral cavity and oropharynx and can occur due to many etiological factors, but smoking and alcohol remain the most common risk factors especially in the Western world6. In South Asian countries, consumption of smokeless tobacco and areca nut products are the main etiological factors associated with OSCC7. Gene mutations may also cause cancer development in the pharynx and oral cavity; however, no specific gene has been identified in OSCCs8. Activation of proto-oncogenes (ras, myc, EGFR) or inhibition of tumor suppressor genes (TB53, pRb, p16) by environmental factors such as smoking, irradiation, and viral infection may increase the risk of oral and oropharynx OSCC9. Most of the oral and oropharynx OSCC cases occur in elderly male patients, with tonsils and tongue being the most commonly affected sites10.\n\nImage is courtesy of Dr. Faraz Kasti (Oral Pathology Division, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia). Written informed consent was obtained from the individual for publication.\n\nIn this review we have briefly described metastasis related to OSCC, some disorders that could transform into OSCC with associated common etiological factors. In addition, a brief account of the diagnosis of OSCC and role of salivary biomarkers in its early detection has also been highlighted. Google Scholar and PubMed search engines were searched with keywords including “oral squamous cell carcinoma”, “OSCC”, “oral cancer”, “potentially malignant disorders in oral cavity”, “etiological factors of OSCC”, “diagnosis of OSCC”, and “salivary biomarkers and OSCC” and our search revealed 500+ results. All the articles in languages other than English and conference abstracts/presentations were excluded. Finally, 77 articles were selected for this study and included in our review.\n\n\nMetastasis\n\nMetastasis could be of two types; regional and/or distant metastasis, as discussed below.\n\nIn terms of regional metastasis, nodal metastasis transpires when tumor cells at the primary site penetrate lymphatic channels and migrate to regional lymph nodes in the neck, forming a micrometastasis11. Lymph node metastasis is a critical prognostic indicator for oral and oropharyngeal carcinomas12. The most common site for OSCC metastasis is cervical lymph nodes, and it reduces the survival rate by 50%13,14. Cancer cells usually spread to the lymph nodes on the same side of the cancer primary site. However, contralateral or bilateral lymph nodes metastasis can rarely occur9. In histopathology, tumor cells dissemination outside the lymph node capsule making the prognosis worse and reducing patient survival rate11. Therefore, a thorough head and neck lymph node inspection and palpation for all first-time patients should be performed to help in early detection of cancer, which will increase the chances for successful treatment and improve prognosis15.\n\nFor distant metastasis, carcinomas require certain biological events in order to spread from the primary tumor site to an anatomically distant site. Several steps are required for cancer cells to spread from their original site to the metastatic one, as shown in the invasion-metastasis cascade16. The cascade starts at the primary tumor site where the cancer cells locally breach the basement membrane to invade the surrounding extracellular matrix and connective tissue17. Then, the tumor cells move to lymphatic or blood vessels and travel to distant metastatic sites. At this point, tumor cells start to extravasate from the vessels into the stroma of the metastatic site18. Initially, tumor cells use the metastatic tissue microenvironment to grow and form micrometastasis. Then, tumor cells expand and colonize to start their own proliferative program and form macroscopic metastasis16. The lung is the commonest site for distant metastasis for head and neck OSCC19. However, metastasis to other organs, such as mediastinal nodes, liver, and bone, have been also reported19,20. Distant metastasis worsens the prognosis and reduces the chances of successful treatment21. Positive regional lymph node involvement, extracapsular invasion of tumor cells, and human papilloma virus negativity are key factors that increase the risk of primary tumor cell dissemination to distant organs20.\n\n\nPotentially malignant disorders (PMDs) transforming into OSCC\n\nEarly detection of cancer is a key factor for improved prognosis and increased patient survival rate. Even though the oral cavity can be easily examined and assessed by direct visual inspection, most OSCC cases are not identified early22. This most likely ensues because patients do not seek dental care on a regular basis and most oral cancers in the early stages are asymptomatic22. Moreover, dentists may not be aware of the different clinical presentations of OSCC and misdiagnose cancers as reactive or benign lesions23. In order to help early discovery and increase the prognosis of cancers, patient awareness about regularly visiting dentists and education of dental practice staff to carefully examine the patients should be raised24.\n\nThere are many PMDs in the oral cavity that have the predisposition to transform into OSCC, a few of which are discussed below in detail.\n\nThe World Health Organization describes “a clinical diagnosis that include any white lesion (plaque or patch) on the oral mucosa that cannot be considered clinically or pathologically as any other disease is a leukoplakia”25. In 1975, Waldron et al. reviewed 3,256 clinical cases defined as “leukoplakia” and found that around 80% of the cases are diagnosed microscopically as either hyperkeratosis or acanthosis26. They also reported that about 17% of the cases were potentially malignant lesions (12.2% mild to moderate dysplasia and 4.5% severe dysplasia or carcinoma in situ) and the diagnosis of OSCC was made in about 3% of the cases that were received with the diagnosis of “leukoplakia”26. Earlier, Bewley and Farwell also reported that OSCC can occur from malignant transformation of leukoplakia27. Therefore, early detection of leukoplakia is key to stop their transformation into aggressive malignant OSCC, which could be hard to treat.\n\nPVL is a destructive form of oral leukoplakia that clinically presents as multiple, slowly spreading white lesions with high reappearance rate and high probability of malignant transformation28. A study of 47 patients diagnosed with PVL showed that around 40% of the patients developed malignant lesions (OSCC or verrucous carcinoma) during follow-up (within 2 years)29. Bagán et al. also reported in their study that there was a high occurrence of patients with PVL developing OSCC in different sites (gingiva and palate being most common)30.\n\nErythroleukoplakia (sometimes called speckled leukoplakia) is a mixed red and white lesion that most likely exhibits more advanced dysplastic changes in histopathological examination compared to leukoplakia31. This lesion usually has irregular margins, and Candida colonization on these lesions is also common32. The chances of speckled leukoplakia for malignant transformation is 18–47%33.\n\nDefined as “Any red lesion of the oral mucosa that cannot be clinically diagnosed as any other condition is called erythroplakia”34. True erythroplakia is a more alarming clinical finding compared to leukoplakia.9 A retrospective study showed that 91% of 58 cases clinically observed as “erythroplakia” were diagnosed as OSCC (51%), carcinoma in situ or severe dysplasia (40%), or mild or moderate epithelial dysplasia (9%)35. Erythroplakia and leukoplakia are usually predecessors of OSCC36 and sometimes also seen adjacent to an OSCC lesion37.\n\nOSMF occurs due to progressive fibrosis of the oral mucosa due to chronic use of areca nut38. Patients diagnosed with OSMF are likely to develop malignant OSCC39. A prospective study was carried out on 371 patients with microscopically proven diagnosis of OSCC and it was reported that around 30% of the patients (112) had a history of OSMF40. However, a study carried out by Chourasia et al. reported an incidence of 4.2% for patients with OSMF transforming to OSCC39.\n\nAn immune-mediated condition that clinically may present as reticular white areas that may or may not be associated with erosive and ulcerative lesions41. There is still debate whether to consider OLP as a PMDs. A previous study in which the data of 20,095 patients was assessed reported 1.1% incidence of OLP patients developing OSCC42. It should be noted however, that erosive type of OLP and patients with history of smoking and alcohol use are likely to suffer from transformation of OLP to OSCC42,43. It was reported in another previous study that tumour recurrence rate of OSCC is higher in patients who had previous OLP than the patients with primary OSCC44.\n\n\nCommon etiological factors of OSCC\n\nVarious etiological factors of OSCC have been reported in the literature. The most common are summarized below.\n\nCigarette smoking helps in the spread of tumors by suppressing immunity and tumor suppressor genes, most importantly p53 and PTEN45. In an earlier study, al-Idrissi reviewed 65 patients with established diagnosis of head and neck OSCC and reported that the majority of these patients were men and 41.5% were smokers46. In another study from China, which included 210 cases, a strong association between long term smoking and OSCC was reported47. Llewelyn and Mitchell from Scotland reported in their study that out of 454 patients with confirmed oral cancer, 60% were smokers and over 95% of those lesions were OSCC48.\n\nA strong connection between drinking alcohol and several cancer types has been described in the literature49. The synergetic effects of alcohol consumption and tobacco smoke increases the risk of OSCC by making the oral epithelium more permeable, dissolving tobacco, and promoting its penetration50. However, chronic use of alcohol alone may lead to OSCC via several mechanisms, including DNA adduct formation, generation of ethanol-related reactive oxygen metabolites, and interference with the DNA-repair mechanism51.\n\nThe consumption of shammah is on the rise in many countries52. It is a combination of powdered smokeless tobacco with ingredients like lime, pepper, ash, and flavoring agents, and people use it by placing it in buccal cavity till the taste penetrates53. In a previous study from Jazan, Saudi Arabia, in which data from 132 patients were recorded, it was reported that the most common cancer detected was OSCC followed by thyroid cancer52. Another study carried out on Yemeni shammah users concluded that there was a strong association between daily shammah usage and formation of leukoplakia (a PMD)54.\n\nKhat is a plant that is mostly used for chewing and is a mixture of cathine and norephidrine55. In a previous study, the prevalence of its consumption was found to be 23.1% among university students of Jazan, Saudi Arabia56. In an earlier case report of one patient, a strong affiliation between khat chewing and growth of OSCC was reported57. Sawair et al. also reported a strong relationship between khat chewing and development of OSCC in their study, which consisted of 649 Yemeni patients58. Lukandu et al. reported from Kenya that chronic khat chewing could lead to abnormal epithelial thickening of oral mucosa and increased keratinization, and fibrosis59.\n\nShisha is commonly available in restaurants, cafes, and other eatery shops in many countries and it contains a high concentration of nicotine, tar, and carbon monoxide60. In water pipe smoking, smoke passes through water and there is a general idea that it is less harmful then cigarette smoking61. In a recently published review, a strong association between water pipe smoking and head and neck cancers was reported62. Zaid et al. reported in a study from Syria and Lebanon that p53 gene mutations were associated water pipe smoking in OSCC63. Al-Amad carried out a study in Jordan, which revealed that 36% of their sample who had oral cancer had a habit of water pipe smoking64.\n\n\nDiagnosis of OSCC\n\nExfoliative cytology is a simple method that could prove useful in early identification of oral cancer as it is based on collection of exfoliated cells for microscopic examination65. It should be noted however that cells can suffer exfoliation normally and/or in the presence of a benign or malignant disease66. Therefore, the most accurate diagnosis of OSCC should only be made by biopsy.\n\nDespite the new diagnostic modalities in oral cancer detection, biopsy and histopathologic analysis remain the gold standard to diagnose OSCC67. An adequate biopsy technique involves local anaesthesia administration, having sufficient width and depth of the excised tissue, correct handling of the tissue, and submission without contamination to aid an accurate definitive diagnosis68.\n\nThe typical diagnosis of OSCC is made by clinical oral examination followed by biopsy of the suspected tissue69. Unfortunately, due to this approach, most OSCC cases either go undetected (at an early stage) or are diagnosed at advanced stages70. In addition, due to late diagnosis, metastasis for OSCC is very common, resulting in a 5-year survival rate of less than 50%71.\n\nHuman saliva could be used for the early detection of various diseases72. OSCC is very common and its early detection can improve the prognosis significantly73. It has been suggested by various researchers that a specific group of protein biomarkers are increased in saliva of individuals with OSCC74. Franzmann et al. reported CD44 as a probable biomarker of head and neck cancer whereas, Nagler et al. described Cyfra-21-1 and cancer antigen-25 to be potential biomarkers for oral cancer74,75. In an earlier study including 395 patients, Elashoff et al. stated an increase in expression of all seven transcriptomes and three proteins as possible markers for OSCC76. They also reported an increase in the levels of IL-8 and subcutaneous adipose tissue in saliva exhibiting maximum levels of sensitivity and specificity to diagnose OSCC77. Similarly, Arellano-Garcia et al. described that expression of IL8 and IL1β were increased in saliva of patients with OSCC as compared with control patients78. Gleber-Netto et al. performed a study involving 180 patients and reported that among the proteomic markers, IL8 and IL1β concentration was greater in OSCC patients when compared with control and dysplasia patients79. Awasthi performed a study that included 64 individuals with diagnosed cases of OSCC, pre-malignant conditions, and healthy controls80. It was revealed from the results of that study that patients with OSCC had increased salivary levels of Cyfra-21-1, lactate dehydrogenase, and total protein concentration in comparison to other groups80.\n\n\nConclusion\n\nOur review concludes that OSCC has the potential for regional as well as distant metastasis. Many PMDs can transform into OSCC with the help of various etiological factors. Diagnosis of OSCC involves traditional biopsy, but salivary biomarkers could also be utilized for its early diagnosis.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "References\n\nCapparuccia L, Tamagnone L: Semaphorin signaling in cancer cells and in cells of the tumor microenvironment--two sides of a coin. J Cell Sci. 2009; 122(Pt 11): 1723–36. PubMed Abstract | Publisher Full Text\n\nJoshi P, Dutta S, Chaturvedi P, et al.: Head and neck cancers in developing countries. Rambam Maimonides Med J. 2014; 5(2): e0009. PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttps://gco.iarc.fr/today/data/factsheets/populations/682-saudi-arabia-fact-sheets.pdf [Accessed: 2nd March, 2020].\n\nAl-Jaber A, Al-Nasser L, El-Metwally A: Epidemiology of oral cancer in Arab countries. Saudi Med J. 2016; 37(3): 249–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTandon P, Dadhich A, Saluja H, et al.: The prevalence of squamous cell carcinoma in different sites of oral cavity at our Rural Health Care Centre in Loni, Maharashtra - a retrospective 10-year study. Contemp Oncol (Pozn). 2017; 21(2): 178–183. 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PubMed Abstract | Publisher Full Text\n\nBagán JV, Murillo J, Poveda R, et al.: Proliferative verrucous leukoplakia: unusual locations of oral squamous cell carcinomas, and field cancerization as shown by the appearance of multiple OSCCs. Oral Oncol. 2004; 40(4): 440–443. PubMed Abstract | Publisher Full Text\n\nNeville BW, Day TA: Oral cancer and precancerous lesions. CA Cancer J Clin. 2002; 52(4): 195–215. PubMed Abstract | Publisher Full Text\n\nWarnakulasuriya S: Clinical features and presentation of oral potentially malignant disorders. Oral Surg Oral Med Oral Pathol Oral Radiol. 2018; 125(6): 582–590. PubMed Abstract | Publisher Full Text\n\nMortazavi H, Baharvand M, Mehdipour M: Oral potentially malignant disorders: an overview of more than 20 entities. J Dent Res Dent Clin Dent Prospects. 2014; 8(1): 6–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYardimci G, Kutlubay Z, Engin B, et al.: Precancerous lesions of oral mucosa. World J Clin Cases. 2014; 2(12): 866–872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShafer WG, Waldron CA: Erythroplakia of the oral cavity. Cancer. 1975; 36(3): 1021–1028. PubMed Abstract | Publisher Full Text\n\nYang SW, Lee YS, Chang LC, et al.: Clinical characteristics of narrow-band imaging of oral erythroplakia and its correlation with pathology. BMC Cancer. 2015; 15: 406. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLapthanasupkul P, Poomsawat S, Punyasingh J: A clinicopathologic study of oral leukoplakia and erythroplakia in a Thai population. Quintessence Int. 2007; 38(8): e448–55. PubMed Abstract | Publisher Full Text\n\nPassi D, Bhanot P, Kacker D, et al.: Oral submucous fibrosis: Newer proposed classification with critical updates in pathogenesis and management strategies. Natl J Maxillofac Surg. 2017; 8(2): 89–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChourasia NR, Borle RM, Vastani A: Concomitant Association of Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma and Incidence of Malignant Transformation of Oral Submucous Fibrosis in a Population of Central India: A Retrospective Study. J Maxillofac Oral Surg. 2015; 14(4): 902–906. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaturvedi P, Vaishampayan SS, Nair S, et al.: Oral squamous cell carcinoma arising in background of oral submucous fibrosis: a clinicopathologically distinct disease. Head Neck. 2013; 35(10): 1404–1409. PubMed Abstract | Publisher Full Text\n\nSaid-Al-Naief N, Rosebush MS, Lynch D: Clinical-pathological conference: case 2. Head Neck Pathol. 2010; 4(3): 221–225. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAghbari SMH, Abushouk AI, Attia A, et al.: Malignant transformation of oral lichen planus and oral lichenoid lesions: A meta-analysis of 20095 patient data. Oral Oncol. 2017; 68: 92–102. PubMed Abstract | Publisher Full Text\n\nMignogna MD, Lo Muzio L, Lo Russo L, et al.: Clinical guidelines in early detection of oral squamous cell carcinoma arising in oral lichen planus: a 5-year experience. Oral Oncol. 2001; 37(3): 262–267. PubMed Abstract | Publisher Full Text\n\nMuñoz AA, Haddad RI, Woo SB, et al.: Behavior of oral squamous cell carcinoma in subjects with prior lichen planus. Otolaryngol Head Neck Surg. 2007; 136(3): 401–404. PubMed Abstract | Publisher Full Text\n\nGandini S, Botteri E, Iodice S, et al.: Tobacco smoking and cancer: a meta-analysis. Int J Cancer. 2008; 122(1): 155–164. PubMed Abstract | Publisher Full Text\n\nal-Idrissi HY: Head and neck cancer in Saudi Arabia: retrospective analysis of 65 patients. J Int Med Res. 1990; 18(6): 515–519. PubMed Abstract | Publisher Full Text\n\nWang X, Xu J, Wang L, et al.: The role of cigarette smoking and alcohol consumption in the differentiation of oral squamous cell carcinoma for the males in China. J Cancer Res Ther. 2015; 11(1): 141–145. PubMed Abstract | Publisher Full Text\n\nLlewelyn J, Mitchell R: Smoking, alcohol and oral cancer in south east Scotland: a 10-year experience. Br J Oral Maxillofac Surg. 1994; 32(3): 146–152. PubMed Abstract | Publisher Full Text\n\nSeitz HK, Becker P: Alcohol metabolism and cancer risk. Alcohol Res Health. 2007; 30(1): 38–47. PubMed Abstract | Free Full Text\n\nFeller L, Chandran R, Khammissa RA, et al.: Alcohol and oral squamous cell carcinoma. SADJ. 2013; 68(4): 176–180. PubMed Abstract\n\nLiu Y, Chen H, Sun Z, et al.: Molecular mechanisms of ethanol-associated oro-esophageal squamous cell carcinoma. Cancer Lett. 2015; 361(2): 164–173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlharbi F: Incidence of head and neck cancers in Jazan province, Saudi Arabia. Saudi J Otorhinolaryngol Head Neck Surg. 2017; 19(2): 47–50. Reference Source\n\nQuadri MF, Alharbi F, Bajonaid AM, et al.: Oral squamous cell carcinoma and associated risk factors in Jazan, Saudi Arabia: a hospital based case control study. Asian Pac J Cancer Prev. 2015; 16(10): 4335–4338. PubMed Abstract | Publisher Full Text\n\nScheifele C, Nassar A, Reichart PA: Prevalence of oral cancer and potentially malignant lesions among shammah users in Yemen. Oral Oncol. 2007; 43(1): 42–50. PubMed Abstract | Publisher Full Text\n\nAl-Hebshi NN, Skaug N: Khat (Catha edulis)-an updated review. Add Biol. 2005; 10(4): 299–307. PubMed Abstract | Publisher Full Text\n\nAlsanosy RM, Mahfouz MS, Gaffar AM: Khat chewing among students of higher education in Jazan region, Saudi Arabia: prevalence, pattern, and related factors. Biomed Res Int. 2013; 2013: 487232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFasanmade A, Kwok E, Newman L: Oral squamous cell carcinoma associated with khat chewing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007; 104(1): e53–e55. PubMed Abstract | Publisher Full Text\n\nSawair FA, Al-Mutwakel A, Al-Eryani K, et al.: High relative frequency of oral squamous cell carcinoma in Yemen: qat and tobacco chewing as its aetiological background. Int J Environ Health Res. 2007; 17(3): 185–195. PubMed Abstract | Publisher Full Text\n\nLukandu OM, Koech LS, Kiarie PN: Oral Lesions Induced by Chronic Khat Use Consist Essentially of Thickened Hyperkeratinized Epithelium. Int J Dent. 2015; 2015: 104812. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShafagoj YA, Mohammed FI: Levels of maximum end-expiratory carbon monoxide and certain cardiovascular parameters following hubble-bubble smoking. Saudi Med J. 2002; 23(8): 953–8. PubMed Abstract\n\nAl Ghobain M, Ahmed A, Abdrabalnabi Z, et al.: Prevalence of and attitudes to waterpipe smoking among Saudi Arabian physicians. East Mediterr Health J. 2018; 24(3): 277–282. PubMed Abstract | Publisher Full Text\n\nPatil S, Awan KH, Arakeri G, et al.: The relationship of \"shisha\" (water pipe) smoking to the risk of head and neck cancer. J Oral Pathol Med. 2019; 48(4): 278–283. PubMed Abstract | Publisher Full Text\n\nZaid K, Azar-Maalouf E, Barakat C, et al.: p53 Overexpression in Oral Mucosa in Relation to Shisha Smoking in Syria and Lebanon. Asian Pac J Cancer Prev. 2018; 19(7): 1879–1882. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Amad SH, Awad MA, Nimri O: Oral cancer in young Jordanians: potential association with frequency of narghile smoking. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014; 118(5): 560–565. PubMed Abstract | Publisher Full Text\n\nGoregen M, Akgul HM, Gundogdu C: The cytomorphological analysis of buccal mucosa cells in smokers. Turk J Med Sci. 2011; 41(2): 205–10. Publisher Full Text\n\nSalih MA, Bushra MO, El Nabi AH, et al.: Comparison between exfoliative cytology and histopathology in detecting oral squamous cell carcinoma. Saudi J Oral Sci. 2017; 4: 46–50. Publisher Full Text\n\nBadvi JA, Kulsoom J, Ujjan IU, et al.: Recent techniques for diagnosis of oral squamous cell carcinoma. EC Microbiology. 2017; 5(5): 165–168. Reference Source\n\nMasthan KM, Sankari SL, Babu NA, et al.: How to help the oral pathologist in making an accurate diagnosis. J Clin Diagn Res. 2013; 7(1): 181–184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFuller C, Camilon R, Nguyen S, et al.: Adjunctive diagnostic techniques for oral lesions of unknown malignant potential: Systematic review with meta-analysis. Head Neck. 2015; 37(5): 755–762. 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}
|
[
{
"id": "61965",
"date": "09 Apr 2020",
"name": "Faisal Aljofi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn etiology section: it has been mentioned that there is a various etiological factors that cause OSCC, most of the articles that have been cited are based on epidemiological statistics. So I think it is better to specify that these are based on epidemiological statistics.\n\nIt has been mentioned that early detection of cancer using certain techniques like salivary bio markers will lead to successful prognosis and treatment, are there any studies showing that there is improved prognosis after early detection? If not then this would be a good way to mention this as a suggestion and future trend to establish studies to show the essential role of early diagnosis.\n\nSuggestions to add diagnostic criteria for early detection of OSCC.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "61967",
"date": "14 Apr 2020",
"name": "Ambreen Rehman",
"expertise": [
"Reviewer Expertise Head and neck oral squamous cell carcinoma",
"micro-vesicles",
"Exosomes",
"oral 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\nThe review on Oral squamous cell carcinoma is a well written comprehensive article. It summarises the aetiology, metastasis and current diagnostic methods of OSCC. It also sheds light on possible use of salivary proteins to aid diagnosis. All the information provided is scientifically valid.\nI would like the author to review the following minor suggested changes:\nRecheck reference 1, I have not found any content related to the incidence of OSCC or Head and neck cancer.\n\nHuman Papilloma Virus (HPV) is one of the leading cause of OSCC. It should be included in the aetiology.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "61966",
"date": "14 Apr 2020",
"name": "Ali Leghari",
"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 article is well written and I would like to accept it. I do have the following suggestions to improve its overall impact.\nIn figure 1: Addition of a clinical picture showing OSCC would benefit the article.\n\nIt would be better if PVL is mentioned as a distinct form of oral leukoplakia and not as a destructive form.\n\nIt would be better if areca nut as a risk factor of OSCC also mentioned separately in heading “Common etiological factor of OSCC”.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-229
|
https://f1000research.com/articles/9-228/v1
|
02 Apr 20
|
{
"type": "Brief Report",
"title": "Composting treatment of fur waste originating from tannery",
"authors": [
"César Puente",
"Janneth Jara-Samaniego",
"Alexander Guapulema",
"Diego Burbano-Salas",
"César Puente",
"Janneth Jara-Samaniego",
"Alexander Guapulema"
],
"abstract": "The tanning industry generates effluents with a high contaminant load represented in a considerable amount of liquid, solid, and gaseous waste. Solid waste, such as residual hair, fat, and meat, are dumped directly in landfills, triggering serious environmental problems. The objective of this research was to compost this waste, previously desulfurized, in a mixture with remnants from pruning as a bulking agent. This low-cost strategy may reduce the total amount of waste. A composting pile was created using residual hair and pruning remnants in a proportion of 3:1 (w/w) and then an inoculum was added. The waste degrading process ran for 141 days, followed by a maturation period of 64 days. Throughout the process, the temperature, moisture, pH, and electrical conductivity (EC) were monitored. The highest temperature reached was 69.7°C, which secured good disinfection. The EC was 0.27 dS/cm, having lower values than Spanish regulations, possibly due to the salt removal over the irrigation periods. The organic matter content decreased continuously, to reach an endpoint of 42%; this result agrees with the high organic matter content of the tannery waste and the pruning remnants. Germination index was 43.55%, which indicates the presence of phytotoxic substances. As compared to the initial value (10.73%) there was partial degrading of these substances. The challenge is to improve this parameter by identifying new mixtures and adding efficient microorganisms that help to degrade phytotoxic substances.",
"keywords": [
"compost",
"tanning",
"waste"
],
"content": "Introduction\n\nModern society has to address a broad suite of sustainability challenges in urban1 and rural areas2,3. The tanning process converts the skins of bovine animals, sheep, and pigs into leather, ready to be used for various purposes. Tannery operations are divided into four processes: riverbank, tanning, post-tanning, and finishing. From an environmental standpoint (cleaner production), the first two processes are important for the volume and pollutant load of effluents, while the last two processes account for the amount of solid wastes and solvent emissions generated in different operations that yield finished leather4. Of the total weight of the skins entering the tannery, 60% is eliminated5. In Tungurahua province, Ecuador, it is estimated that out of the 920,000 cattle headcount, 500,000 skins are treated in tanneries yearly6.\n\nThis study proposes, through a desulfurization process, to reduce the sulfide concentration in these residues, and then take advantage of their nutrients (organic matter and nitrogen) to carry out a composting process. As a result, a stabilized product, compost, will be obtained. In order to improve the C/N ratio, the pellet residues will be mixed with pruning remnants. Specifically, this study addresses the following objectives: (i) characterize the fur waste, (ii) build and test a desulfurization system for the fur waste, (iii) identify the appropriate percentage of fur and pruning remnants, (iv) establish the appropriate composting system, and (v) determine the quality of the compost.\n\n\nMethods\n\nA total of 576 kg of fur residues was obtained from the tannery “EL ALCE leathers” located in Guano canton, Chimborazo province.\n\nDesulfurization consisted of catalytic oxidation of sulfide containing residues with compressed air and magnesium sulfate (ALMACEN EL AGRO, Riobamba-Ecuador), 0.4 g/(kg of pellet of fur). Sulfur determination was carried out according to APHA 4500-S2F protocol7. Duration of the operation was 5 h in accordance with the protocol7. With this process, the removal of sulfur was accomplished by converting it into sulfuric acid and reducing sulfur dioxide emissions.\n\nPhysical-chemical analysis enabled the evaluation of chemical oxygen demand (COD) and sulfide concentration in fur residues (meat, grease, and hair) and of fur water. We determined COD according to APHA 5220 D protocol8.\n\nPreviously desulfurized fur residues were dried and crushed (to a size ranging from 1 cm to 5 cm) and mixed with crushed pruning remnants according to CCIA 20139. pH and electrical conductivity were measured for solid/water ratio of 1:10 (w/w) at each turning using glass electrode (Model Seven2Go Advanced Single-Channel Portable pH Meter, Mettler, Toledo). Organic matter was determined by calcination at 430°C, according to the method described in Navarro et al.10. Humidity was measured gravimetrically in an oven at 105 °C for two hours to constant weight. Phosphorus was determined by spectrophotometer (UV5, Mettler Toledo, USA) at 440 nm according to AOAC 958.02/960.02 protocol11. Potassium was measured by flame photometry according to AOAC 965.09/945.04 protocol12. Total nitrogen was determined according to AOAC 955.04 protocol13.\n\nIn order to accelerate the composting process, an inoculum was prepared with a mixture of mature compost (2.5 lb.), chicken residue (2.5 lb.), pork slurry (10 L), and molasses (2.5 L) coming from collection center of the Escuela Superior Politécnica de Chimborazo. With the conditioned waste, a 2.5 m × 2.5 m × 1.5 m pile was assembled. Three parts of fur were used for one part of pruning remnants; the homogeneous mixture was placed into a 100-liter tank for fermentation for 24 hours.\n\nIn the initial and final samples (after 2 months of maturing), in addition to the parameters listed above, germination index (GI) were determined according to Moreno and Moral 201114, using radish seeds (Raphanus sativus) (supplier: El Agro Riobamba-Ecuador) to establish the maturity of the obtained compost; briefly: GI is calculated from the ratio of germinated seeds and the root length of radish seeds in an aqueous compost solution14.\n\n\nResults and discussion\n\nSulfides were reduced by 23.7% (Table 1). This percentage could increase by extending the duration of the desulfurization process.\n\nThe waste degradation process lasted for 141 days, followed by a maturity of 64 days. The stack showed a reduction in volume, size, and odor, indicative of the decomposition of the materials used, including residual hair, as previously observed by Numpaque and Viteri15. One important parameter to highlight is the rapid increase in temperature, reaching maximum values of 69.7°C (Figure 1), which ensured a good disinfection process16.\n\nOrange: average temperature (°C) in the compost. Blue: average temperature (°C) of environment.\n\nThe moisture content varied from 45 to 65%, which is considered as optimal17. We found slightly alkaline medium, with an average final pH 7.94. The amount of organic matter (Figure 2) decreased to 42%, and this data is consistent with the Spanish Standard Royal Decree 506/201318 on fertilizer products. Contents of total N (3.62%), P (0.31%), and K (0.39%) of the matured compost were higher than those obtained in composting processes of waste materials with wastewater from the tannery industry, which present values of 1.36%, 0.001%, and 0.23%, respectively19. Reported electrical conductivity (EC) values of 0.27 dS/cm were below the Spanish Standard18; this is possibly due to the salts washing off over irrigation periods. It is preferable for the compost to have low EC values, since the presence of salts in high concentrations inhibits plant growth20.\n\nTo establish the maturity of the compost, the GI value was determined using Radish seeds (Raphanus sativus). In the final compost, GI was 43.55%, a value that indicates the presence of phytotoxic substances. Nevertheless, if we start from the assumption that GI of the pile at zero time has been 10.73%, a clear decrease of these substances is evident. The increase in GI is an indication of the degree of maturity of the compost21.\n\n\nConclusions\n\nThe quantity and type of nutrients in this compost allowed the rapid growth of microorganisms, while recording thermophilic temperatures close to 70°C, which guaranteed an adequate hygiene of the product. Based on the contents of organic matter, in accordance with the Spanish Standard Royal Decree 506/201318, it was assessed that the obtained compost belonged to Group 6. Organic amendments, No. 2, Annex 4 ‘Waste of Leather Industries, of the Skin and Textile’5, indicates that with an adequate control of the composting process, parameters of an acceptable product can be obtained, in relation to the pollutant load. Considering that the initial germination index was 10.73% and the final 43.55%, it can be concluded that there was a partial elimination of phytotoxic substances. The challenge is to improve this parameter by looking for new mixtures and inoculating efficient microorganisms that allow these substances to be degraded. At present, the compost obtained could be used in restoration projects of deteriorated and soils poor in organic matter, or as cover material for sanitary landfills.\n\n\nData availability\n\nFigshare: Avarage Pile temperature, https://doi.org/10.6084/m9.figshare.11753187.v122.\n\nFigshare: Physical Chemica, Seeds, https://doi.org/10.6084/m9.figshare.11755182.v123.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nElmqvist T, Andersson E, Frantzeskaki N, et al.: Sustainability and resilience for transformation in the urban century. Nat Sustain. 2019; 2: 267–273. Publisher Full Text\n\nSándor Z, Tállai M, Kincses I, et al.: Effect of various soil cultivation methods on some microbial soil properties. DRC Sustainable Future. 2020: 1(1): 14–20. Publisher Full Text\n\nJakab A: The ammonium lactate soluble potassium and phosphorus content of the soils of north-east Hungary region: a quantifying study. DRC Sustainable Future. 2020; 1(1): 7–13. Publisher Full Text\n\nSalas G: Eliminación de sulfuros por oxidación en el tratamiento del agua residual de una curtiembre. Rev Per Quim Ing Quim. 2005; 8(1): 49–54. Reference Source\n\nCorredor J: El residuo líquido de las curtiembres estudio de caso: cuenca alta del Río Bogotá. Ciencia e Ingeniería Neogranadina. 2006; 16(2). Publisher Full Text\n\nZurita G: Análisis descriptivo de la sustentabilidad económica, social y ambiental de la industria del cuero del Ecuador. En: consulta: enero 2018. 2016. Reference Source\n\nAPHA 4500-S2-F: Iodometric Method. Standard Methods 21st Edition. National Environmental Methods Index. 2005. Reference Source\n\nAPHA 5220 D. Reference Source\n\nCorporación Colombiana de Investigación Agropecuaria CCIA, Corpoica: Producción de Abonos Organicos de Buena Calidad. Bogota: Produmedios. 2013: 80–81. Reference Source\n\nNavarro AF, Roig A, Cegarra J, et al.: Relationships between total organic carbon and oxidable carbon in calcareous soils. Commun Soil Sci Plant Anal. 1993; 24(17–18): 2203–2212. Publisher Full Text\n\nAOAC 958.02/960.02: Official Methods of Analysis. 21st Edition. Reference Source\n\nAOAC 965.09/945.04 NTC 1369:2009: reference: AOAC. 18th Edition.\n\nAOAC 955.04: Official Methods of Analysis. 21st Edition.\n\nMoreno JG, Moral R: Compostaje. [aut. libro] Moreno J. y Moral R. Compostaje. Madrid (España) : Ediciones Paraninfo S.A., 2011; 99–100. Reference Source\n\nNumpaque V, Viteri S: Biotransformación del pelo residual de curtiembres. Rev Cienc Agr. 2016; 33(2): 95–105. Publisher Full Text\n\nRoman P, Martinez MY, Pantoja A: Manual de compostaje del agricultor. FAO. 2013. Reference Source\n\nHaug R: The practical handbook of compost engineering. Lewis publishers. 1993. Publisher Full Text\n\nStandard Royal Decree 506/2013. Reference Source\n\nÍñiguez G, Rodríguez R, Virgen G: Compostaje de material de descarne y aguas residuales de la industria de curtiduría. Rev Int Contam Ambient. 2006; 22(3): 113–123. Reference Source\n\nMasaguer A, Benito M: Evaluación de la calidad del compost. En: Compostaje. (eds.= Moreno, J., Moral, R. Ed. Mundi-Prensa, Madrid. 2008; 570.\n\nVarnero M, María T, Rojas A, et al.: ÍNdices de Fitotoxicidad en Residuos OrgÁnicos Durante el Compostaje. Suelo Nutr Veg. [online]. 2007; 7(1): 28–37. Publisher Full Text\n\nBurbano: Avarage Pile temperature. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11753187.v1\n\nBurbano: Physical Chemica, Seeds. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11755182.v1"
}
|
[
{
"id": "62425",
"date": "24 Apr 2020",
"name": "Imre Vágó",
"expertise": [
"Reviewer Expertise Alternative (and also traditional) plant nutrition methods",
"parameters influencing the effectivity of plant nutrients on different soil types",
"plant cultures",
"water supply. Effects of different plant nutrients on the quantity and quality of plant products",
"furthermore on the chemical and some microbial parameters of soils. Effects of global warming and changing distribution of precipitation on the methods of sustainable plant 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\nI read with great interest the article entitled “Composting treatment of fur waste originating from tannery” written by César Puente, Janneth Jara-Samaniego, Alexander Guapulema, and Diego Burbano-Salas in which:\nauthors draw attention on the importance of process industrial waste coming from tanneries;\n\na 141 days (waste degradation) + 64 days (maturing) long compost experiment is described. The study was carried out with the purpose of discuss a low-cost strategy to decompose tanning industrial waste.\n\nI have found that the paper contains interesting and up-to-date information on the topic about waste management. In that sense, I find that the research finds well within the scope of the journal, and adds new information. However, there are a series of limitations that make me recommend some minor revisions of this manuscript.\nMajor concerns:\nIntroduction should be expanded.\n\nThe study propones a comparison between the results obtained at different time points in the composting process, namely: COD, sulfur, Organic matter %. It should be mentioned that only Organic matter was monitored from the mentioned parameters. COD and sulfur values shown at the beginning and at the end of compost process only. It would be more fortunate to compare these parameters. If only at two time points - these parameters (COD and sulfur) were measured, then Organic matter values should be selected at same time points for comparison purposes, if enough data available for further statistical tests.\n\nAuthors concluded that “…Considering that the initial germination index was 10.73% and the final 43.55%, it can be concluded that there was a partial elimination of phytotoxic substances. The challenge is to improve this parameter by looking for new mixtures and inoculating efficient microorganisms that allow these substances to be degraded.” What missing are the authors’ recommendations regarding how to improve germination index according to the observations discussed in the study. Please specify which exact parameters must to be “improved” e.g. in pH value, N ?! etc. in order to improve the germination index.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": []
},
{
"id": "85498",
"date": "26 Aug 2021",
"name": "Linee Goswami",
"expertise": [
"Reviewer Expertise Environmental Biotechnology",
"Bioremediation",
"Waste management"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript deals with a very relevant problem. However, the introduction needs elaboration. In the last decade, several works on \"composting and vermicomposting\" of tannery waste have been reported. Researchers have worked extensively. Therefore, more research citations are needed. The work needs to highlight the novelty of the work as compared to all other works done with tannery wastes.\n\nThe study design needs to address a few more parameters in order to be inclusive. Results section needs elaboration. How the compost treatments affecting the growth parameters of radish seeds require proper data or figures to corroborate the findings. Microbial growth also needs to be presented with some quantitative or qualitative assessments. Distinction between beneficial or pathogenic can be the first step. Or, authors can just analyse NFB and PSB population growth following in-vitro methods.\n\nDesulphurization can only be a part of the physicochemical analysis.\nFur industry tannery waste also contains a high amount of Cr-salt / Na ions in the chemical spill. That needs to be verified and compared among the treatment combinations.\nThe manuscript does not mention statistical analysis. For better understanding and acceptability among the readers, the authors can perform some basic statistical analysis of the data provided, report the t-test or variability among the treatment combinations. A time dependent study can be more comprehensive with proper statistical data. It can be improved using SPSS/ Prism/R.\n\nThe source data underlying the results seems reproducible.\n\nWith the current results, I think it supports the conclusion. However, as mentioned earlier, addition of some physicochemical parameters will provide the reader a better visibility.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-228
|
https://f1000research.com/articles/9-227/v1
|
02 Apr 20
|
{
"type": "Brief Report",
"title": "Innovative strategies to fight antimicrobial resistance: crowdsourcing to expand medical training",
"authors": [
"Eneyi E. Kpokiri",
"Jehan Z. Budak",
"Christina C. Chang",
"Jason J. Ong",
"Claude Mabilat",
"Rosanna W. Peeling",
"David Van Duin",
"Joseph D. Tucker",
"Jehan Z. Budak",
"Christina C. Chang",
"Jason J. Ong",
"Claude Mabilat",
"Rosanna W. Peeling",
"David Van Duin",
"Joseph D. Tucker"
],
"abstract": "Background: Antimicrobial resistance is a serious public health concern across the world, but public awareness is low, few educational resources on diagnostics exist and professional interest in infectious diseases is waning. To spur interest in infectious disease, emphasize the role of diagnostics in management of resistant infections and develop educational resources to support antimicrobial stewardship. Methods: We employed crowdsourcing methods, using an open challenge contest to solicit clinical cases on antimicrobial resistance and clinical diagnostics. Results: We received 25 clinical cases from nine countries. After screening, 23 cases were eligible for judging. Three cases emerged as the top finalists and were further developed into an open access learning module on diagnostics and antimicrobial resistance. Conclusions: Crowdsourcing methods are beneficial for generating interest in infectious disease and developing educational resources to support antibiotic stewardship.",
"keywords": [
"Antimicrobial resistance",
"clinical diagnostics",
"crowdsourcing",
"infectious diseases"
],
"content": "Introduction\n\nAntimicrobial resistance (AMR) is a major public health threat. Reports from the World Health Organisation (WHO) global surveillance and Global Point Prevalence Survey show high rates of inappropriate antibiotic use1, suggesting the need for enhanced antimicrobial stewardship. Yet public awareness of AMR remains low2 and the pipeline from medical school into careers relating to infectious diseases is weak. The numbers of US trainees entering infectious diseases decreased 41% between 2009 and 20173.\n\nThere is a need to respond to the lack of interest in infectious disease training through various methods, including expanding medical training on infectious diseases3. Most medical schools give limited teaching on AMR and even less on the use of diagnostics to reduce inappropriate use of antibiotics4. A European study reported that in all but one of seven medical schools studied, the majority of students wanted further education on antibiotic prescribing, which is an essential aspect of AMR management5. A US study also suggested that physicians receive inadequate training on the interpretation of antibiograms6. Other studies from the UK7, China8 and Ethiopia9, suggest the need for more AMR and diagnostics content in medical curricula. The WHO recently highlighted the importance of undergraduate training in prudent prescribing and research highlights the need for more AMR and diagnostics training for healthcare workers around the world10.\n\nIn response to this need, we organised a crowdsourcing project, soliciting clinical cases on diagnostics/AMR from medical students and physicians. Crowdsourcing is a bottom-up approach that allows many individuals to attempt to solve a problem and then shares solutions with the public11. Crowdsourcing contests typically convene a steering committee, engage citizens or a particular group to participate, evaluate entries, recognize finalists and share solutions with the public (Table 1). It has been used in medical research and piloted as a tool to develop medical education materials12.\n\nWHO- World Health Organisation, AMR- Antimicrobial Resistance, ID-Infectious Disease\n\nOur crowdsourcing approach focused on developing an educational resource for medical students and trainees on infectious disease diagnostics/AMR. The overall goal of the challenge contest was to spur enthusiasm for infectious diseases and increase infectious disease diagnostics/AMR knowledge to support antibiotic stewardship. The aim of the call was to encourage medical students, trainees, physicians, and others to collect or write clinical cases to develop educational materials for AMR.\n\n\nMethods\n\nWe set up a global steering committee comprised of 19 individuals from six WHO regions (seven women and 12 men). The steering committee included experts in laboratory science, medicine, public health, health communications, and medical education. Members were selected based on sex, geographic region, and expertise. We accepted clinical cases focused on used of diagnostics in AMR from mid-March until May 1, 2018. This call was disseminated through social media channels (including Facebook, Twitter and YouTube), partner organizations, professional association mailing lists, and in-person events. The call for participation was translated into the six official languages of the WHO, but only entries in English were accepted. Links to a one-minute video and a website developed for the contest were shared as part of the call. The website received an average of 117 page views each week. Banners and posters (see Extended data)13 were also printed and displayed in some hospitals and medical schools in London to create awareness about the contest. All cases were screened for eligibility by two steering committee members and then sent to four physician judges for evaluation using pre-specified criteria. Judges included infectious disease fellows and residents identified by the steering committee. Eligibility criteria published alongside details of the open contest included a focus on AMR clinical case, written in English, using less than 2000 words, and included at least one image. Participants were also required to obtain consent from patients to share cases. The selected judges were neither part of the steering committee nor involved in the design of the call. At this stage, each case was scored between 0 and 10 by the judges. Criteria set for judging the cases include focus on diagnostics and AMR, relevance to medical teaching and capacity to enhance appropriate antibiotic use. The judging rubric is available as Extended data13.\n\nIndividual scores were collated, and the mean score for each case was calculated. At the end of the judging process, the finalists (cases that achieved a mean score of 7/10 or greater) were announced. These clinical cases had a second level of review from three independent judges with detailed feedback on specific areas to improve overall quality and understanding of the cases presented.\n\nAll case authors were provided commendation certificates for participation. The finalists were provided with individualized feedback and supported to attend a multidisciplinary diagnostics/AMR focused symposium in London. Finalist cases were disseminated through Partners ID Images. Partners ID Images is an open access online library focused on infectious diseases.\n\nWe used a modified Delphi method as part of a one-day symposium to identify and prioritize key learning objectives for the AMR learning module that was developed using finalists cases14. The Delphi survey was included as part of activities in a one-day AMR symposium held in London on November 1, 2018, which had experts in AMR research and practice in attendance. The symposium had a line up different AMR research presentations and a panel discussion on diagnostics and AMR. A total of 30 participants, including physicians, medical microbiologists, clinical researchers and medical post graduate students, who attended the one-day AMR symposium and were asked to participate in a survey. Participants were briefed about the survey contents and were also informed that participation was voluntary. The initial round in the morning session consisted of 30 participants and the second round in the afternoon had 21 participants, as some participants attended only the morning session. After two rounds, there was consensus to include 12 objectives (see Extended data, supplementary file 3) in the AMR learning module.\n\nA crowd voting platform was set up on Partners ID images for the general public to select a crowd favourite from the three finalists. The voting page was open for two weeks and received 334 votes, mostly from the US, Peru, Australia and the UK. A random number generator was used to select 17 voters (5% of total votes) to receive a free digital Sanford Guide app.\n\n\nResults\n\nWe received 25 clinical case entries from nine countries. Peru had the highest number of entries – seven (28%), followed by the US with six entries (17%). Other submissions were received from Nigeria (n=4), China (n=3), Australia (n=1), Canada (n=1), Ethiopia (n=1), Paraguay (n=1), and Zambia (n=1). After screening, 23 cases were eligible for the next stage of judging (see Underlying data13); two entries were excluded for not being relevant to the subject of the call. In total, 19 were original cases written by participants, and four were adapted from already published literature. All cases were submitted alongside a signed declaration of patients’ consent from the authors. The cases described clinical presentations of drug resistant organisms and the role of diagnostics in their management. An overview is given on the SESH Global website.\n\nOut of the 23 eligible cases, three cases emerged as finalists with mean scores of seven or above. Most cases achieved scores greater than five; only six entries (26%) had mean scores less than five. Underlying data shows scores for each case13.\n\nThe crowd favourite was a clinical case on pyelonephritis caused by metallo-beta-lactamases producing Pseudomonas aeruginosa. All three finalist cases (see Extended data, Supplementary file 1)13 were further reviewed with medical education experts from partner organizations and integrated into an online diagnostics/AMR interactive learning module (see Extended data, Supplementary file 2)13. This was done with the help of the results of the Delphi approach (see Underlying data for results)13.\n\n\nDiscussion\n\nCrowdsourcing provides several advantages in the development of medical training materials compared to conventional approaches, including duration of time needed, cost, and global coverage. Our challenge was able to solicit over 20 cases in less than two months, faster than most medical education development. The total cost of the challenge was less than having experts develop clinical cases. Finally, the global composition of our steering committee and case contributors allowed us to develop a resource that may be relevant in diverse global settings.\n\nHowever, our crowdsourcing contest had several limitations that warrant consideration. This contest received fewer clinical cases than other crowdsourcing contests. The low number of cases may have been related to the lack of official partnerships with a larger conference or professional associations, the relatively high requirements – including need to obtain patient’s consent – in the guidelines for submissions, and the timing of the call. Our call for cases coincided with a similar call for clinical cases on infectious diseases from the Infectious Diseases Society of America. Despite the overall low number of submissions, we had sufficiently strong cases to create the learning module and develop new case content for the database.\n\nOne gap identified during the challenge was the limited strategic priorities within AMR medical education. The purpose of the learning module is to increase awareness and understanding of diagnostics and AMR in adult and paediatric medicine. This could be used as Continuing Medical Education (CME) or integrated into undergraduate medical curricula.\n\nWhile we have created an open access learning resource on diagnostics and AMR (supplementary file 2), there is still a need for more educational resources related to AMR. Crowdsourcing may be a useful adjunct in the development of medical education materials. Crowdsourcing could be used in a variety of settings to encourage interest in infectious diseases, decrease unnecessary antibiotic use and promote antibiotic stewardship.\n\n\nData availability\n\nOpen Science Framework: Innovative strategies to fight antimicrobial resistance: crowdsourcing to expand medical training. https://doi.org/10.17605/OSF.IO/WVP9G13.\n\nThis project contains the following underlying data:\n\nAMR_Delphi_ResultsTable. (Results of the two-part Delphi study).\n\nClinical cases (The 23 clinical cases submitted).\n\nScores for AMR Clinical cases. (Judges scores for each submitted case).\n\nAMR_Delphi[1]. (Raw results from Delphi study round 1).\n\nAMR_Round2[1]. (Raw results from Delphi study round 2).\n\nOpen Science Framework: Innovative strategies to fight antimicrobial resistance: crowdsourcing to expand medical training. https://doi.org/10.17605/OSF.IO/WVP9G13.\n\nThis project contains the following extended data:\n\nSupplemental_digital_appendix_1FINALIST_Clinical_cases[1]. (Supplementary file 1: Summaries of the case finalists).\n\nSupplemental_digital_appendix__2_The_learning_module[1] (Supplementary file 2: How to access the diagnostic/AMR learning module)\n\nSupplemental digital appendix 3 The learning Objectives. (Supplementary file 3: Learning objectives).\n\nAMR contest flyer (flyer used to advertise AMR case competition)\n\nAMR Contest roll up banner (banner used to advertise AMR case competition)\n\nCrowdvotingAnnouncement14Oct2018 (announcement made to encourage crowdvoting)\n\nAMRsurvey_ToPrint[1] (blank survey used for Delphi study round 1)\n\nAMRsurvey_round2[1] (blank survey used for Delphi study round 2)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from each of the patients.",
"appendix": "Acknowledgements\n\nThe authors wish to acknowledge the support of Mark Miller, Isabelle Canaiux, Gros Marie-Francoise, Brian Schwartz, and Rajesh Tim Gandhi. The diagnostics/AMR challenge contest Steering Committee, the London School of Hygiene and Tropical Medicine International Diagnostics Centre, University of California San Francisco, Partners ID Images (https://www.idimages.org), Social Entrepreneurship to Spur Health (SESH Global) and the authors of our clinical cases.\n\n\nReferences\n\nVersporten A, Zarb P, Caniaux I, et al.: Antimicrobial consumption and resistance in adult hospital inpatients in 53 countries: results of an internet-based global point prevalence survey. Lancet Glob Health. 2018; 6(6): e619–e29. PubMed Abstract | Publisher Full Text\n\nMason T, Trochez C, Thomas R, et al.: Knowledge and awareness of the general public and perception of pharmacists about antibiotic resistance. BMC Public Health. 2018; 18(1): 711. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonura EM, Armstrong WS: Increasing Subspecialization in the Field of Infectious Diseases: Evaluating Challenges and Strategies to Move Forward. J Infect Dis. 2017; 216(suppl_5): S594–S9. PubMed Abstract | Publisher Full Text\n\nAbbo LM, Cosgrove SE, Pottinger PS, et al.: Medical students' perceptions and knowledge about antimicrobial stewardship: how are we educating our future prescribers? Clin Infect Dis. 2013; 57(5): 631–8. PubMed Abstract | Publisher Full Text\n\nDyar OJ, Pulcini C, Howard P, et al.: European medical students: a first multicentre study of knowledge, attitudes and perceptions of antibiotic prescribing and antibiotic resistance. J Antimicrob Chemother. 2014; 69(3): 842–6. PubMed Abstract | Publisher Full Text\n\nNand P, Wilson MD, Cohen SH, et al.: Curbing antimicrobial resistance: Do physicians receive adequate education about antibiograms? J Infect. 2016; 72(1): 127–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastro-Sanchez E, Farrell S, Drumright L, et al.: Do we need to review and escalate the antimicrobial stewardship education in health and veterinary undergraduate courses in the United Kingdom? Results of a national survey. Int J Infect Dis. 2014; 21: 203. Publisher Full Text\n\nHu YH, Wang XM, Tucker JD, et al.: Knowledge, Attitude, and Practice with Respect to Antibiotic Use among Chinese Medical Students: A Multicentre Cross-Sectional Study. Int J Environ Res Public Health. 2018; 15(6): pii: E1165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeid MA, Hussen MS: Knowledge and attitude towards antimicrobial resistance among final year undergraduate paramedical students at University of Gondar, Ethiopia. BMC Infect Dis. 2018; 18(1): 312. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers Van Katwyk S, Jones SL, Hoffman SJ: Mapping educational opportunities for healthcare workers on antimicrobial resistance and stewardship around the world. Hum Resour Health. 2018; 16(1): 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLendvay TS, White L, Kowalewski T: Crowdsourcing to Assess Surgical Skill. JAMA Surg. 2015; 150(11): 1086–7. PubMed Abstract | Publisher Full Text\n\nTucker JD, Meyers K, Best J, et al.: The HepTestContest: a global innovation contest to identify approaches to hepatitis B and C testing. BMC Infect Dis. 2017; 17(Suppl 1): 701. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKpokiri EE: Innovative strategies to fight antimicrobial resistance: crowdsourcing to expand medical training. 2020. http://www.doi.org/10.17605/OSF.IO/WVP9G\n\nBudak J, Kpokiri EE, Abdoler E, et al.: 2547. Prioritizing Antimicrobial Resistance Learning Objectives Through a Modified, Two-Round, One-Day Delphi at a Multidisciplinary Conference. Open Forum Infectious Disease. 2019; 6(Supplement_2): S885. Publisher Full Text"
}
|
[
{
"id": "77504",
"date": "25 Jan 2021",
"name": "Chijioke Oliver Agomo",
"expertise": [
"Reviewer Expertise Pharmacy practice",
"public health",
"and health & social sciences."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 for this review. The authors clearly stated the aims of their research. In addition, they selected appropriate methodology & design for the study, to address the aims and objectives of their research.\nHowever, I have provided a few suggestions below:\nIntroduction:\nIn second paragraph, you mentioned: “There is a need to respond to the lack of interest in infectious disease training through various methods…” Could there exist other reasons for this lack of interest in infectious disease, for example, the fear of contracting and then dying from infectious diseases, etc.?\nMethods:\nDid you seek an approval from any ethics committee for this study? If so, please state this in your paper.\nYou said, “The call for participation was translated into the six official languages of the WHO, but only entries in English were accepted.” Was there any particular reason why the call for participation was translated into six official languages, but acceptance for entries was only in English? Why do you think this was the most appropriate method to use for this study, and how can this be justified? To what extent did this affect the final selection of entries and the final results? These could be reflected in your limitations, as well as discussing any potential bias. Also, what did you do enhance the credibility of your results?\nDelphi approach: please provide readers with a brief description of the Delphi method & process. What advantage does the Delphi have over the other methods such as brainstorming, analytical hierarchy process, data-intensive decision making, etc.? On the same paragraph: please change, “…AMR symposium and were asked to participate in a survey.” to, “…AMR symposium were asked to participate in a survey.”\nCrow voting: here you stated that, “A random number generator was used to select 17 voters (5% of total votes) to receive a free digital Sanford Guide app.” How did you arrive at that decision to select only 5% of votes, instead of the entire 100%?\nResults/discussion\nPlease include more information about the contribution of your study to existing knowledge in AMR, policy and wider practice. Provide a specific recommendation(s) for further studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": []
},
{
"id": "82948",
"date": "26 Apr 2021",
"name": "Joshua T Ravensdale",
"expertise": [
"Reviewer Expertise Molecular Microbiology",
"Antimicrobial Resistance",
"Medical Microbiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of the manuscript describe a crowdsourcing approach to identifying best practice in antimicrobial therapy and infectious disease care to develop new training tools for infectious disease specialists. The design of the study is sound and the aims of the study are very relevant considering the threat of AMR in pathogens and the spread of COVID-19 globally.\nThe main issue I have with the study is that the results and discussion are lacking in detail and do not give the reader a full idea of the relevance of the results. For the most part the writing is fine, however, there are some grammatical errors that should be addressed. Unfortunately, as the manuscript was not provided to me with each line numbered I could not provide the authors with specific corrections. However, I strongly suggest the authors re-read the manuscript and make corrections where needed as some sentences were difficult to understand due to poor grammar and/or sentence structure.\nAbstract; I would say that interest in infectious diseases and diagnostics is at the forefront of the publics mind thanks to the COVID-19 pandemic.\nLast sentence in the background section does not address the purpose of the study.\nThe results section doesn’t really give the reader an informed idea of what was found in the study besides saying that a new learning module was created. Likewise, the conclusion is also vague and doesn’t explain how these results are novel.\nIntroduction; 2nd last sentence of 1st paragraph: Describing the pipeline as “weak” does not give the reader a clear idea what the author is referring to.\nMethods; 4th sentence of 1st paragraph: “Used diagnostics” does not make sense in this context.\nResults; Please provide more information on how the top three studies were scored the highest. Was it just based off the criteria described in the methodology or were there aspects of the cases that were of particular merit? As identifying these traits seems to be one of the main aims of this study, it may be useful to include more specifics of the cases in the results section.\nDiscussion; In the first paragraph no references are provided to support the authors statements that the crowdsourcing approach is quicker and cheaper than other methods.\nThe discussion focused on the limitations of the study but did not describe the relevance of the results of the study. For example, it would be insightful for the reader to know how the authors used the data to develop their learning module, how best practice methodologies were identified and how these compared with standard treatment, how the data could be used to encourage more medical graduates to specialise in infectious diseases and the relevance of the project during the COVID-19 pandemic. It would also be interesting to discuss how the crowdsourcing model could be used to identify inappropriate, ineffective, or harmful practices being used by the medical community to draw attention to areas in medical training that need to improve.\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? 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/9-227
|
https://f1000research.com/articles/9-25/v1
|
16 Jan 20
|
{
"type": "Study Protocol",
"title": "Burden and risk factors for snakebite in India: protocol for a systematic review",
"authors": [
"Soumyadeep Bhaumik",
"Robyn Norton",
"Jagnoor Jagnoor",
"Robyn Norton",
"Jagnoor Jagnoor"
],
"abstract": "Introduction: Snakebites are a neglected tropical disease with a high burden in South and South-East Asia and sub-Saharan Africa. In 2019, the World Health Organization (WHO) released a roadmap which aims for a 50% reduction in death and disability due to snakebite globally by 2030. It is estimated that India has the highest number of snakebite deaths in the world. Objective: To synthesize evidence on the burden (incidence/ prevalence, mortality, morbidity, health facility and economic), and risk factors for snakebite in India. Methods: We will search for peer-reviewed literature and grey literature in six electronic databases (MEDLINE, EMBASE, Global Health, PsychInfo, CENTRAL, SafetyLit) and hand-search IndMed, conference abstracts, relevant websites and citation tracking. Two reviewers will screen and extract data independently with a third reviewer acting as an arbiter for any inconsistencies. Quality of the included studies will be assessed using the Joanna Briggs Institute (JBI) critical appraisal tools. For burden, data from facility based and community-based studies will be synthesised and reported separately, except in the case of studies conducted concurrently. We will conduct narrative analyses with the aim of understanding patterns in data through tabulation for both burden and risk factors evidence synthesis. The PROGRESS Plus lens will be used to explore equity pertaining to burden of snakebites. Analyses for each individual risk factor-outcome pair will be conducted and reported separately. If appropriate, meta-analyses will be conducted as per JBI guidelines, assessing heterogeneity using Tau-squared, Cochran’s Q test and Chi-squared (p > 0.05) tests. We plan to conduct sub-group analyses based on setting, study design, sex/gender, age-groups, tribal people and occupation. A funnel plot will be generated if there are more than nine studies included in a specific meta-analysis, to assess publication bias. Asymmetry of the funnel plot will be adjudged using the Egger, Begg and Harbord tests.",
"keywords": [
"Snake Bites",
"Epidemiology",
"India",
"Prevalence",
"Incidence",
"risk factor",
"Health systems",
"economic costs"
],
"content": "Background\n\nSnakebites are a neglected tropical disease, with considerable burden in South Asia, Southeast Asia, and sub-Saharan Africa1. They are known to affect rural, indigenous and economically disempowered communities who lack political voice2–4. A modelling study using data on venomous snake distribution, health-care access, and availability of snake anti-venom, estimated that globally 146.70 million people live in snakebite prone areas lacking quality health-care provisions5. However, broad consensus is that these numbers are underestimates as many affected by snakebite are ‘out-of-reach’ of the formal health systems6,7. Snakebite envenomation also causes long-term health effects, and is believed to have high social and economic impacts in affected communities6,8,9. Morbidity and socio-economic impact of snakebite is not well understood and remains under-researched globally9,10.\n\nIn 2018, recognising the public health impact of snakebite on vulnerable communities the World Health Assembly (WHA) passed a resolution to address the burden of snakebite4. Earlier in 2019 the World Health Organization (WHO) released a roadmap which aims to halve by 2030 the death and disability due to snakebite globally11. The WHO strategy rests on four pillars of action: empowering and engaging communities; ensuring safe, effective treatment; strengthening health systems; and increasing partnerships, coordination and resource usage through collaborations11.\n\nMore than a third of the global deaths, about 46,000 annually, are estimated to occur in India12 with not much known about other aspects of burden7 or risk factors in the country. Understanding the epidemiology of snakebites (in terms of incidence/prevalence of bites and envenoming, mortality, morbidity and risk factors) at the national and subnational level together with economic costs and health facility burden is critical for developing strategies, plans and programs to address the burden of snakebite. There are no systematic reviews on the burden and risk factors for snakebite in India, although evidence synthesis on burden and impact has been done for other countries or regions13–16. The current article provides the protocol for a systematic review on the burden and risk factors for snakebite in India.\n\n\nObjectives\n\nTo synthesize evidence on the burden (incidence/ prevalence, mortality, morbidity, health facility and economic), and risk factors for snakebite in India\n\n\nResearch questions\n\n1. What is the burden (incidence/ prevalence, mortality, morbidity, health facility, and economic) of snakebite in India nationally and sub-nationally?\n\n2. What are the risk factors related to snakebite (bite, envenomation, death, adverse outcomes) in India?\n\n\nProtocol and registration\n\nThe objectives, inclusion criteria and methods of analysis for this systematic review are specified in advance and documented in this a priori protocol.\n\n\nEligibility criteria\n\nThe systematic review consists of two distinct evidence syntheses - burden (incidence/prevalence, mortality, morbidity, health facility and economic); and risk factors for snakebite (bite, envenomation, death, adverse outcomes). Synthesis of evidence for each domain will be conducted and reported separately in alignment with recent Cochrane guidelines17.\n\nWe will include studies that meet the following criteria:\n\n• Population – involving human participants from India, irrespective of age, gender or any other characteristics.\n\n• Condition – snakebite irrespective of how it is diagnosed, measured or confirmed.\n\n• Setting - facility or community-based studies; autopsy-based studies will be included for understanding aspects of burden, as relevant.\n\n• Burden Outcomes –\n\n○ Incidence/ prevalence– incidence rate of snakebite or snakebite envenoming (i.e. clinical envenoming) (population or age-specific) from community-based studies only; prevalence rate of snakebite or snakebite envenoming from community-based, autopsy-based and facility-based studies;\n\n○ Mortality – incidence death rate (mortality rates per 100,000) due to snakebite (population or age-specific) from community-based studies only; case fatality rate due to snakebite from facility-based studies.\n\n○ Morbidity – measured using any validated disability or quality of life tools or DALYs or any other standardised measure (as defined by the authors) community and facility-based studies.\n\n○ Health facility burden- measured in terms of proportions and/or percentages for the following:\n\n▪ Visits/admissions in emergency department, clinic/out-patient department, in-patient department (for both venomous and non-venomous bites)\n\n▪ Days of inpatient admission (for both venomous and non-venomous bites)\n\n▪ Requirement of specialist consultation\n\n▪ Requirement for referral in higher facility\n\n▪ Requirement of ventilatory support / dialysis support / blood transfusion in acute setting (as defined by primary study authors)\n\n▪ Requirement of fasciotomy to manage compartment syndrome (as defined by primary study authors)\n\n▪ Requirement of long-term rehabilitation support (as defined by primary study authors)\n\n○ Economic burden- from provider perspective or client perspective (direct and/or indirect costs) – as defined and measured by primary study authors.\n\n○ Study design –\n\n○ cohort studies (prospective or retrospective)\n\n○ cross-sectional studies (analytical)\n\n• There will be no restriction by year of publication or language.\n\nWe will include studies that meet the following criteria:\n\nPopulation – involving human participants with snakebite or at-risk of snakebite from India. We will not include hospital based forensic- autopsy studies for understanding risk factors.\n\nSetting: facility or community-based studies; autopsy-based studies will be excluded as they cannot give data on risk factors.\n\nRisk factors of interest and related outcomes -No a priori list of risk factors is listed as the scope of the evidence synthesis is broad. We will include any risk factor related to following outcomes\n\n○ incidence of snakebite, snakebite envenomation (clinical illness), death due to snakebite from community-based studies (reported in terms of relative risks (RR), odds ratios (OR), hazard ratios (HR), standardized incidence ratios (SIR) or a standardized mortality ratios (SMR); adjusted or otherwise)\n\n○ snakebite envenomation (clinical illness), death due to snakebite, adverse outcome (as defined by study authors) from facility-based studies (reported in terms of relative risks (RR), odds ratios (OR), hazard ratios (HR), standardized incidence ratios (SIR) or a standardized mortality ratios (SMR); adjusted or otherwise)\n\nStudy design –We will include the following study designs:\n\n○ cohort studies (prospective and retrospective)\n\n○ case-control studies\n\n○ cross-sectional studies (analytical)\n\nWe will not include risk-modelling studies as they are not within the scope of the current evidence synthesis.\n\nThere will be no restriction by year of publication or language.\n\n\nInformation sources and search strategy\n\nWe will search the following electronic databases for eligible studies using adaptions of the MEDLINE search strategy developed for this purpose (see extended data18):\n\nMEDLINE\n\nEMBASE\n\nGlobal Health\n\nPsychInfo\n\nCENTRAL\n\nSafetyLit\n\nWe will hand-search IndMed (a bibliographic database covering prominent peer reviewed Indian biomedical journals), conference abstracts (including but not limited to Indian Public Health Association Conference - IPHACON, Annual Conference of the Toxinological Society of India- TSICON, Annual National Conference of Indian Society Of Toxicology - TOXOCON: as available) and contact researchers of repute in India to identify more studies. We will also hand search vital statistics data, government reports, population surveys or white papers which have reported on the burden and/or risk factors for snakebite specifically in relevant websites. We will also hand search the reference lists of all included studies found by other methods to retrieve additional records.\n\n\nStudy selection\n\nTwo review authors will independently assess the eligibility of primary studies based on titles and/or abstracts in the first phase. We will then acquire the full text of all papers identified as potentially relevant by at least one review author. Two review authors will then assess these papers independently and classify them into four categories – included for burden; included for risk factors; included for both burden and risk factors; excluded. We will resolve disagreements, by discussion with a third reviewer acting as an arbiter. We will attempt to contact study authors for further information, if necessary.\n\n\nData management\n\nWe will extract data using a standardised data extraction protocol, developed by adding extra data elements to the JBI recommended minimum standards for data extraction for prevalence, incidence and risk factor systematic reviews19,20. Data management will be done using the Joanna Briggs Institute- The System for the Unified Management, Assessment and Review of Information (SUMARI).\n\n\nQuality of included studies\n\nWe will appraise the quality of the included studies by using the JBI quality assessment tools for cohort, analytical cross-sectional and case-control studies19,20.\n\n\nSynthesis of results\n\nData from facility based and community-based studies will be synthesised and reported separately, except in the case of studies which have conducted both concurrently.\n\nWe will narratively summarise the results of the study. An equity lens will be applied to understand incidence/prevalence, mortality and morbidity equity issues in a granular fashion. We will use the PROGRESS plus framework21 for this purpose and extract and synthesise disaggregated data, if available on the framework parameters (PROGRESS-Plus - Place of residence; Race/tribal people; Occupation; Gender/sex; Religion; Education; Socioeconomic status; Social capital; and “Plus” to indicate other possible equity factors which might affect the outcomes of interest in relation to snakebite). We will assess patterns in the data through tabulation of results.\n\nWe do not intend to conduct a meta-analysis or any additional quantitative analysis and will present data as reported. In general heterogeneity between studies on prevalence and incidence is known to be common, rendering meta-analysis inappropriate19. In addition, snakebite as a condition is known to be localised in nature. As such, pooling of data from heterogenous studies into one pooled estimate will not reflect the variability in the burden of the condition at sub-national and local levels. The phenomenon of diluting the burden of snakebite by pooling of specific local data into national snakebite incident rate data has been previously recognised and been described as the 'tyranny of mean values’6.\n\nAnalysis for each individual risk-factor outcome pair will be conducted separately. If appropriate, meta-analyses will be conducted as per the JBI Guidelines20. A random or fixed effects model with a 95% CI will be chosen for the meta-analysis based on the presence of heterogeneity assessed by Tau-squared, Cochran’s Q test and Chi-squared (p > 0.05) tests20. We plan to conduct sub-group analyses for the following, if enough studies are found:\n\nStudy design\n\nSetting (community based; facility based)\n\nSex/gender (male; female; other)\n\nAge groups: Children (less than 10 years), adolescent (11–19 years), young adults (20–24 years)\n\nTribal / non-tribal people\n\nOccupation (agricultural/plantation workers or farmers, and fishermen)\n\nSensitivity analyses will be conducted, as appropriate, and if enough studies are available, to assess robustness of results but we are not specifying any a priori sensitivity analyses in the protocol phase. We will generate a funnel plot to assess publication bias if there are more than nine studies included in a specific meta-analysis. Funnel plot asymmetry will be tested by statistical tests (Egger test, Begg test, Harbord test) as appropriate.\n\nWe will publish the results of this review and will make the data accessible openly in re-usable format. The data will also be disseminated through evidence summaries and policy briefs to stakeholders in governments, public institutions and communities.\n\nThe search, screening and subsequent steps will be undertaken after the protocol completes peer-review.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended Data Set : Burden and risk factors for snakebite in India: protocol for a systematic review. https://doi.org/10.6084/m9.figshare.11536776.v118\n\nThis project contains the following extended data:\n\nSearch strategy snakebite obj1.pdf (Study search strategy)\n\nPRISMA-P checklist for ‘Burden and risk factors for snakebite in India: protocol for a systematic review’. https://doi.org/10.6084/m9.figshare.11536776.v118\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nKasturiratne A, Wickremasinghe AR, de Silva N, et al.: The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 2008; 5(11): e218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGutiérrez JM: Current challenges for confronting the public health problem of snakebite envenoming in Central America. J Venom Anim Toxins Incl Trop Dis. 2014; 20(1): 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHabib AG: Public health aspects of snakebite care in West Africa: perspectives from Nigeria. J Venom Anim Toxins Incl Trop Dis. 2013; 19(1): 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSachan D: The snake in the room: snakebite’s huge death toll demands a global response. BMJ. 2018; 361: k2449. Publisher Full Text\n\nLongbottom J, Shearer FM, Devine M, et al.: Vulnerability to snakebite envenoming: a global mapping of hotspots. Lancet. 2018; 392(10148): 673–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRepublic of Costa Rica: Recommendation for the Adoption of an Additional Disease as a Neglected Tropical Disease : The Case for Snakebite Envenoming. Geneva: WHO. 2017. Reference Source\n\nBhaumik S: Snakebite: a forgotten problem. BMJ. 2013; 346: f628. PubMed Abstract | Publisher Full Text\n\nWilliams DJ, Faiz MA, Abela-Ridder B, et al.: Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming. PLoS Negl Trop Dis. 2019; 13(2): e0007059. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrison RA, Gutierrez JM: Priority Actions and Progress to Substantially and Sustainably Reduce the Mortality, Morbidity and Socioeconomic Burden of Tropical Snakebite. Toxins (Basel). 2016; 8(12): pii: E351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGutiérrez JM, Burnouf T, Harrison RA, et al.: A Call for Incorporating Social Research in the Global Struggle against Snakebite. PLoS Negl Trop Dis. 2015; 9(9): e0003960. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Snakebite envenoming: A strategy for prevention and control. Geneva: World Health Organization. 2019. Reference Source\n\nMohapatra B, Warrell DA, Suraweera W, et al.: Snakebite mortality in India: a nationally representative mortality survey. PLoS Negl Trop Dis. 2011; 5(4): e1018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTochie JN, Temgoua MN, Njim T, et al.: The neglected burden of snakebites in Cameroon: a review of the epidemiology, management and public health challenges. BMC Res Notes. 2017; 10(1): 405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdiriweera DS, Diggle PJ, Kasturiratne A, et al.: Evaluating temporal patterns of snakebite in Sri Lanka: the potential for higher snakebite burdens with climate change. Int J Epidemiol. 2018; 47(6): 2049–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeitosa ES, Sampaio V, Sachett J, et al.: Snakebites as a largely neglected problem in the Brazilian Amazon: highlights of the epidemiological trends in the State of Amazonas. Rev Soc Bras Med Trop. 2015; 48: 34–41. PubMed Abstract | Publisher Full Text\n\nChippaux JP: Epidemiology of snakebites in Europe: a systematic review of the literature. Toxicon. 2012; 59(1): 86–99. PubMed Abstract | Publisher Full Text\n\nThomas J, Kneale D, McKenzie JE, et al.: Determining the scope of the review and the questions it will address. Cochrane Handbook for Systematic Reviews of Interventions. 2019; 13–31. Publisher Full Text\n\nBhaumik S, Norton R, Jagnoor J: Extended Data Set: Burden and risk factors for snakebite in India: protocol for a systematic review. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11536776.v1\n\nMunn Z, Moola S, Lisy K, et al.: Chapter 5: Systematic reviews of prevalence and incidence. In: Aromataris E MZ, ed. Joanna Briggs Institute Reviewer's Manual: Joanna Briggs Institute. 2017. Reference Source\n\nMoola S, Munn Z, Tufanaru C, et al.: Chapter 7: Systematic reviews of etiology and risk. In: Aromataris E MZ, ed. Joanna Briggs Institute Reviewer's Manual: Joanna Briggs Institute. 2017. Reference Source\n\nO'Neill J, Tabish H, Welch V, et al.: Applying an equity lens to interventions: using PROGRESS ensures consideration of socially stratifying factors to illuminate inequities in health. J Clin Epidemiol. 2014; 67(1): 56–64. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "59189",
"date": "04 Feb 2020",
"name": "Bert Avau",
"expertise": [
"Reviewer Expertise Evidence synthesis methodology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this systematic review protocol aim to map the burden and risk factors for snakebite in the Indian context. To do so, they have composed two separate review questions: one on the incidence and prevalence of snakebite, the other one on the risk factors that may contribute to the burden of snakebite. A systematic review of the existing literature seems an appropriate method to reach their objectives. The authors have reported their work appropriately according to the PRISMA-P checklist. However, several suggestions for improving/clarifying the reporting of their work are made below, especially regarding synthesizing their findings.\nThe authors include as datasets a completed PRISMA-P checklist and a search string to search for paper in Ovid Medline. These datasets are in my opinion accessible and useable.\nSpecific points for consideration regarding the reporting of the methods provided:\n1) Page 4 - Eligibility criteria for risk factors for snakebite in India:Can you please elaborate as to why risk modelling studies are not within scope of this systematic review? The scope of this review is as I understood broad, summarizing all the evidence on burden and risk factors for snakebite. To my understanding, risk modelling studies may contribute to the scope as set by the authors.\n2) Dataset search strategy: I would search for \"envenom*\" instead of \"envenomation*\". This will make sure you also find records that use the term \"snake envenoming\" instead of \"snake envenomation\".\n3) Page 5 - Synthesis methods for evidence synthesis on the burden of snakebite in India: Though I agree it may be misleading to pool data from different diverse regions into one national summary, it may be worthwhile to consider whether combining findings from studies from the same (or similar) regions is possible. Therefore I’m not sure whether a priori excluding the possibility of a meta-analysis for incidence/prevalence data is the best option here.\n4) Page 5 - Synthesis methods for evidence synthesis of risk factors for snakebite in India: It is generally not advisable to choose the type of meta-analysis method (fixed or random) a posteriori, based on the observed level of heterogeneity (JBI Handbook section 5.5.8.2 & Cochrane Handbook section 10.10.4.11). Rather, one should consider the underlying assumptions of the two models and decide a priori which method would likely be the most appropriate in relation to the expected type of data. Please address this issue.\n5) Page 5 - Synthesis of results: In case meta-analyses are considered inappropriate, one will need to synthesize the data in another way. Please provide a method for doing this. The guidance by Campbell et al.2 and Chapter 12 of the Cochrane Handbook3 may provide valuable input for this.\n6) Page 5 - Synthesis of results: In case a meta-analysis can be performed, it is not unlikely that you will encounter statistically heterogeneous results. Please elaborate on how you will assess heterogeneity (i.e. what will you consider a heterogenous result?). Guidance on this can be found in Chapter 10.10 of the Cochrane Handbook4.\n7) PRISMA-P Item 12: Although the authors state “a standardised data extraction protocol, developed by adding extra data elements to the JBI recommended minimum standards for data extraction for prevalence, incidence and risk factor systematic reviews” will be used, it is not clear which items the authors will extract. It would be transparent to add an empty copy of this data extraction sheet, to be clear about which data items will be extracted.\n8) PRISMA-P Item 14: The authors need to clarify how they will use the risk of bias assessment during data synthesis.\n9) PRISMA-P Item 17: I disagree that assessment of the body of evidence is not applicable. Several studies will contribute to outcomes, therefore it is useful to assess our overall confidence in the evidence gathered from different studies. I’m not saying the authors should definitely use the GRADE approach for this, but being transparent in how the overall strength of the body of evidence will be evaluated is necessary.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "5314",
"date": "02 Apr 2020",
"name": "Soumyadeep Bhaumik",
"role": "Author Response",
"response": "We thank the reviewer for the comments on the scope and appropriate methods as well as the suggestions for improvement and clarification has been useful to us for revising the protocol (numbers in response correspond to numbers in query . 1. We thank the reviewer for consideration of inclusion of risk modelling studies. We have followed the guidelines of JBI (Joanna Briggs Institute) for systematic reviews of risk. The guidelines recommend study designs and we have included them : https://wiki.joannabriggs.org/display/MANUAL/7.2.1+Observational+Study+Designs . Risk modelling studies are not recommended study design for inclusion. A risk modelling study is a statistical procedure for assigning a probability of developing a future outcome. We contend that this is not within the purview of this evidence synthesis as risk modelling studies look into prediction in contrast to cohort or case-control studies. 2. We have now modified the search strategy in the dataset. 3. Thank you for acknowledging that a national pooling might be misleading. We have had considerable discussion on the issue within the team and now looked at the issue of burden being meta-analysed more granularly and made substantial changes to reflect this in the manuscript. The summary of the new analysis for synthesising evidence is provided: • We conceptualised burden to go beyond incidence or mortality, to mean the following which are reflected as outcomes: incidence/prevalence, mortality, morbidity, health facility and economic. Data from these come from either community based or facility based or autopsy-based studies. • For incidence/prevalence, mortality and morbidity we will pool data at state level and conduct meta-analysis (if appropriate) in a state-wise fashion (political borders as per Surveyor General of India – May 2020) from community-based studies only. We will not pool data from any facility based studies as data from them will be dependent on patient , health facility and catchment area characteristics implying considerable clinical heterogeneity • We will not pool data from health facility and economic outcomes and conduct meta-analysis for the same reasons as above. We have provided more information on what will be done when meta-analysis is not envisaged subsequently. 4. We have now elaborated on the section to reflect changes in alignment with suggestion. In summary, we have amended to align with the JBI recommendation of using the random effects model a priori. We will use a fixed-effect approach only if we assess heterogeneity (clinical and methodological and statistical) to be minimal. 5. We have earlier mentioned on Page 5 that we will assess patterns in the data through tabulation of results based on JBI guidelines. We have now taken note of the new Cochrane Handbook and added additional information on analysis methods where meta-analysis is not possible or not envisaged .It is revised to now include information about the structured reporting of results guidelines provided by the reviewer and following approaches will be taken as appropriate for different outcomes. A summary is provide below : • Summarizing effect estimates to report range and distribution of observed values • Vote counting based on direction of effect (with on comment on the magnitude of effect) using harvest plot and/or effect direction plot. • We will use visualisation tools, as appropriate (harvest plot, effect direction plot, etc). We have also elaborated on sub-group analysis being done for the same as relevant for different analysis. 6. We have now elaborated on this section to clarify that for the purpose of the review will be consider > 40% heterogeneity to be a “heterogenous result” and have detailed methods used for the same 7. We have further elaborated to mention that the data extraction form will be pilot tested in the first few studies. Providing an empty data extraction form is not required as per PRISMA-P guidelines. We have now put information on the “planned method of extracting data from reports (such as piloting forms, done independently, in duplicate), any processes for obtaining and confirming data from investigators” as required in PRISMA-P. We reiterate and as noted in out transparency statement, all data will be provided with the systematic review findings. 8. We have clarified further on the use of sensitivity analysis if meta-analysis and how the use the risk of bias assessments will be used during the data synthesis process. 9. We agree that using GRADE to assess overall confidence in evidence would be useful, however GRADE is not an appropriate tool for evidence synthesis for burden and risk factors. GRADE is suitable for interventions and diagnostic test accuracy studies only and GRADECerQUAL for qualitative studies. To the best of our knowledge there are no appropriate approach available for our purpose, for which consensus tool exist. PRISMA-P is a reporting guideline and not a methodological guidance and we have reported that overall strength of body of evidence will not be assessed in our research and rendering corresponding section in PRISMA-P not applicable."
}
]
},
{
"id": "58690",
"date": "17 Feb 2020",
"name": "Abul M. Faiz",
"expertise": [
"Reviewer Expertise Clinician and Toxicologist"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe objective related to Burden and risk factors are clumped with incidence/prevalence, mortality and morbidity and envenomation; may be provided in more elaboration specially risk factor because adverse outcome and death is not risk factor for envenomation. The authors have reported their work appropriately according to the PRISMA-P checklist.\n\nEligibility criteria: It should be made clear that all or part of the eligibility criteria will need to be fulfilled. Risk factor is not properly addressed here and authors has no plan for risk modelling. Risk variables are not clear too. I think more emphasis was given on burden. So more clarification and clear objective about risk factor should be mentioned.\n\nCondition: ‘irrespective of how it was diagnosed’- is it, that venomous and non venomous bites will be grouped together which will cause problem in interpretation of the results. Dataset search strategy: It can also include venomous and antivenom as we consider economic burden .\n\nSynthesis of result: It is not unusual to get heterogeneous result due to regional variability and low impact article. So how the authors analyse this heterogeneity should be mentioned.\n\nHealth facility burden (page 3): Usually all/most of the patients of snakebite present at the emergency department unless manifested as chronic condition. Definition/clarity is required on ‘specialist’, ‘higher facility’, ‘compartmental syndrome’, ‘long term rehabilitation support’. How uniformity among the investigators on decision for ventilatory support, dialysis, blood transfusion will be interpreted. How the individual component of data will be collected.\nStudy design (page 4): It would not be wise to have a study having no restriction of ‘year of publication’ to see the burden and risk factors which may change over time.\n\nRisk factors: a list may be developed. 2nd bullet is not clear- snakebite irrespective of envenomation may also be admitted, so admission does not necessarily mean envenomation. Searching other sources: how the authors will identify ‘researchers of repute’.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "5315",
"date": "02 Apr 2020",
"name": "Soumyadeep Bhaumik",
"role": "Author Response",
"response": "We thank the reviewer for the very useful comments and would like to clarify that we already have two separate review questions and corresponding sections. We elaborate concerns below : Burden is conceptualised as incidence/prevalence, mortality, morbidity, health facility and economic Risk factors- We agree with the concern about the risk factor and clarify and in lieu of reviewer feedback have revised to include the following two only and removed adverse outcome and envenomation as suggested: Risk factor for a snakebite- this contributed to preventive aspects of bite itself Risk factor for death due to snakebite Based on the comments, we have reworded the section to provide more clarity. Eligibility criteria: We have revised the manuscript, to provide more clarity on the eligibility criteria. Risk factor: We have clarified the objectives for the risk factors as above. We clarify that we do not intend to or plan to do risk modelling as this is a systematic review and not a risk modelling study. Risk modelling cannot be done with summary statistics available form published literature. We have detailed methods for meta-analysis. Condition: We thank the reviewer for the comment. We have considered this already and since we are conducting a systematic review of published studies, we must use diagnostic definitions as used by primary study authors. Hence the use of irrespective. Primary study authors would classify venomous and non-venomous and their definitions will be used. Dataset search strategy : We have revised the search strategy to include key words related to snake anti-venom as per reviewer comments. The keywords added in search are : “anti-venom* or antivenom* or anti-dote* or antidote* or anti-snake or antisnake” We have not included venomous in the search strategy as it leads to decrease in the sensitivity of the search leading to retrieval of many studies on venomous animals including snake . These are not on snakebites. Any study which has looked at snakebite burden and risk factor will have the word “bite” and will be retrieved from this current search strategy. The changes are reflected in the dataset. Synthesis of result: We have now elaborated on this section to clarify that for the purpose of the review will be consider > 40% heterogeneity to be heterogenous Health facility burden : We agree to the observation that most bites present at emergency department but we do not want to be exclude those who visit clinic/out-patient department, in-patient department, so as to comprehensively report on health system burden, inclusive of for disability/chronic condition resulting from snake bite. Definition/clarity is required on ‘specialist’, ‘higher facility’, ‘compartmental syndrome’, ‘long term rehabilitation support’: We thank the reviewer but since it is a systematic review of already conducted primary studies, we are going to use the definition as per primary study authors. We cannot control how individual component of data is collected- this may or may not be uniform, but we will collect and report the different definitions /modalities of measurement along with the outcome data. We chose to put no time restrictions, so as to report change over time. Risk factors : We have not made an a priori list as we are looking to review all risk factors. This is also not required as this is not a primary study and out search strategy has been made broad to look for all studies on snakebite in India. We have included admission as part of burden. We conceptualised this as health system burden since both venomous and non-venomous bites need evaluation in a health facility and is hence contributing to health system burden. As mentioned, we are collecting this data for both venomous and non-venomous bites and these will be reported separately. Searching other sources: We will contact experts as per knowledge of the review team- this is a standard evidence synthesis approach. Detailed number of experts who contributed to identification of newer studies will be provided at full review phase."
}
]
}
] | 1
|
https://f1000research.com/articles/9-25
|
https://f1000research.com/articles/9-176/v1
|
10 Mar 20
|
{
"type": "Method Article",
"title": "First derivative ATR-FTIR spectroscopic method as a green tool for the quantitative determination of diclofenac sodium tablets",
"authors": [
"Khairi M. S. Fahelelbom",
"Abdullah Saleh",
"Ramez Mansour",
"Sadik Sayed",
"Abdullah Saleh",
"Ramez Mansour",
"Sadik Sayed"
],
"abstract": "Background: Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is a rapid quantitative method which has been applied for pharmaceutical analysis. This work describes the utility of first derivative ATR-FTIR spectroscopy in the quantitative determination of diclofenac sodium tablets. Methods: This analytical quantitative technique depends on a first derivative measurement of the area of infrared bands corresponding to the CO stretching range of 1550-1605 cm-1. The specificity, linearity, detection limits, precision and accuracy of the calibration curve, the infrared analysis and data manipulation were determined in order to validate the method. The statistical results were compared with other methods for the quantification of diclofenac sodium. Results: The excipients in the commercial tablet preparation did not interfere with the assay. Excellent linearity was found for the drug concentrations in the range 0.2 – 1.5 w/w %. (r2= 0.9994). Precision of the method was assessed by the repeated analysis of diclofenac sodium tablets; the results obtained showed small standard deviation and relative standard deviation values, which indicates that the method is quite precise. The high percentage of recovery of diclofenac sodium tablets (99.81, 101.54 and 99.41%) demonstrate the compliance of the obtained recoveries with the pharmacopeial percent recovery. The small limit of detection and limit of quantification values (0.0528 and 0.1599 w/w %, respectively) obtained by this method indicate the high sensitivity of the method. Conclusions: First derivative ATR-FTIR spectroscopy showed high accuracy and precision, is considered as nondestructive, green, low cost and rapid, and can be applied easily for the pharmaceutical quantitative determination of diclofenac sodium tablet formulations.",
"keywords": [
"Diclofenac sodium tablet",
"FTIR spectroscopy",
"infrared quantitative analysis",
"first derivative",
"method development"
],
"content": "Introduction\n\nDiclofenac sodium (DS) is a nonsteroidal anti-inflammatory drug (NSAID) that is known for its potent pharmacologic activity. It is analgesic and also ameliorates acute and subchronic inflammation. The drug plays a unique dual inhibitory role simultaneously on cyclo-oxygenase (COX) and lipoxygenase enzymes1. The drug is widely marketed under a variety of generic and brand names and is available in several dosage forms. The chemical structure of DS is shown in Figure 1, the IUPAC nomenclature is 2-[(2,6 dichlorophenyl)aminophenyl]-acetic acid sodium salt.\n\nChemical formula of diclofenac sodium.\n\nSeveral quantitative analytical methods have been implemented to test the active pharmaceutical ingredient (API) of DS in its various marketed dosage forms. These quantitative approaches include high performance liquid chromatography (HPLC)2–4, gas chromatography5, UV–visible spectrophotometry6, spectrofluorometry7, densitometry8, potentiometry8,9, and Raman spectroscopy10,11. The HPLC methods were also employed by the official US and British pharmacopeias for the analysis of DS12,13.\n\nHowever, until recently, few reports have investigated and explored the capability and feasibility of infrared spectroscopy in its quantitative approach as a potential alternative to the aforementioned classic procedures14. This work aims to expand on previous reports exploring quantitative Fourier transform infrared (FTIR) approaches to quantitatively determine the APIs in a number of DS tablet dosage forms.\n\nTraditionally, infrared spectroscopy has been extensively used in a qualitative manner. Elucidation of chemical composition has usually been achieved by analyzing incident radiation absorptions at a specific wavelength. Hence, the technique proves to be a powerful detector of functional groups. Each functional group is known to have its own distinguishable IR signature.\n\nDuring the last few decades, there has been an exponential growth in the quantitative applications of IR spectroscopy15.\n\nThe technique has proved to be an appealing alternative to examine DS APIs in terms of quantitative outcomes. The results of numerous reports have indicated that FTIR offers unparalleled advantages over other techniques, which makes it a versatile alternative. The technique is inherently fast and covers a wide wavenumber range. It is also non-destructive and high resolved spectra can be obtained for almost all types of samples16. Simultaneous analysis of sample matrices does not necessary require special sample preparation using challenging and lengthy procedures17. The technique is environmentally friendly as a result of the lack of any need for hazardous solvents or reagents18. The quality of results are comparable to most powerful techniques and the cost of routine analysis is extremely economical19.\n\nPublished research in this regard relies on the attenuated total reflection (ATR) sampling technique of modern FTIR spectrometers. ATR allows recording of an FTIR spectrum of solid and liquid samples directly without any further preparations20. Nevertheless, the sampling of DS still requires pressing an accurately measured amount of the drug or its commercial dosage into a potassium bromide (KBr) disc to recode perfect spectra. Herein, the work retested the technique and expanded its capabilities by examining the direct spectral recording using a powder of DS (and its commercial formulations) mixed with KBr samples21. The new proposal simplifies the use of the ATR-FTIR technique tremendously. The exclusion of KBr disk sampling in each run will ultimately improve the procedure in terms of the time that the overall runs require. Consequently, modern FTIR instruments are affordable and this work proves powder mixtures or thin film samples produce comparable results, implying the unnecessity of KBr discs or press machines.\n\nThe evolution of quantitative capabilities of FTIR techniques may be attributable to the vast instrumentation advancements associated with powerful computers. Additionally, sampling techniques including flow analysis (FA) and ATR have been revolutionized to allow for analysis of almost any sample type - solids, liquids, solutions, gases and vapors - directly.\n\nQuantitative FTIR has been successfully implemented in several areas of industrial pharmacy. It has been evolving as the potential technique of quality control protocols, in particular, analysis of APIs in a broad spectrum of pharmaceutical dosage forms and formulations14. The technique’s inherent characteristics and nature bears unequivocally bright prospects. It is considered a purely green analytical chemistry technique. It is fast, easy to operate by a moderately experienced technician, can analyze any sample with little to no preparation, covers a wide range of spectrums to analyze most pharmaceutical products, has high resolution and is nondestructive. Importantly, it is environmentally friendly since no solvent or harmful reagents are required for the complete analysis22.\n\nThis work also demonstrates and utilizes modern spectrometers’ powerful function of the first derivative spectra. The function provided the basic tool that allows the spectrometer resolution to be tuned to the degree required to greatly enhance the band separation of each spectrum. In this work, the ATR-FTIR spectrum of the pure DS was recorded. The powerful data processing and acquisition software associated with the spectrometer allowed the first derivative spectra to be obtained. The first derivative spectra indicated the degree of IR band overlapping within each spectrum. The selection of the IR band that best correlates with the concentration of DS without any interference of other bands is based solely on the first derivative spectra.\n\n\nMethods\n\nIR spectroscopic-grade potassium bromide was checked prior to usage through the loss on drying method based on the recommended British Pharmacopia (BP) procedure13, which involves introducing 1g of the sample into a dry bottle, reheating and reweighing until a constant weight is obtained.\n\nReference DS chemical standard was provided by Neopharma Pharmaceutical Manufacturing, Abu Dhabi, UAE. Three different DS commercial tablets were obtained from local pharmacies in Al Ain city, UAE, namely: Rumafen (diclofenac sodium 50 mg); Olfen TM 50 (diclofenac sodium 50 mg); and Diclogesic 50 (diclofenac sodium 50 mg).\n\nThe FTIR instrument IRAffinity-1 CE (Shimadzu®, Kyoto, Japan), equipped with MiracleTM Single Reflection Horizontal ATR Accessory (Pike® Technologies) and IResolution Software (Shimadzu, version 1.60; Parameters: Measurement mode: % transmittance; Apodization: Happ-Genzel; No of scans: 15; Resolution: 16.0; Range (cm-1): Min 700, Max 2000), was used for the screening and quantitative analysis in this study. Non-proprietary spreadsheet processing software could be used to analyze the raw data obtained. The samples were dried in an oven (WiseVen®, Won-032, S. Korea) and an analytical balance (AUW220D, Shimadzu®) was used for all weights.\n\nA calibration curve was prepared from six different DS standard concentrations within the range of 0.2–1.0 % w/w. An appropriate quantity of DS was diluted with potassium bromide to get each concentration and was thoroughly ground in a mortar for 10 min to ensure sample homogeneity. Infrared spectra of each measurement were converted to the first derivative spectra. The area under the curve (AUC) of each calibration standard was measured in the range 1550–1605 cm-1. This band corresponds to the CO stretching of carboxylic sodium salts.\n\nTo determine the API content for the commercial tablets in this study, ten tablets were accurately and individually weighed, then the whole sample was finely powdered for each brand. Samples were prepared by mixing and thoroughly grinding an appropriate quantity of each tablet powder with potassium bromide to get 0.5% w/w of DS. 100 mg of the mixture was transferred to the diamond ATR top plate of the spectrometer over the diamond crystal. IR measurements were run in triplicate and the average was calculated for each run.\n\nSPSS version 21 (SPSS Inc., Chicago, IL, USA) was used for descriptive and analytical statistics.\n\n\nResults and discussion\n\nThe proposed ART-FTIR analytical method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines23.\n\nValidation of the method included the following parameters: linearity, selectivity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness.\n\nThe proportional relationship between the AUC and concentration was evaluated by constructing a linear regression of five concentrations (0.2, 0.4, 0.6, 0.8, 1%). The excellent linearity obtained was indicated by the correlation coefficient value r2 = 0.9994. Table 1 shows the data analysis of the DS calibration curve and the linear relationship is presented in Figure 3a and 3b.\n\nLOD, limit of detection; LOQ, limit of quantification.\n\nSelectivity is used to justify the ability of the method to accurately quantify the existence of DS in the presence of other pharmaceutical additives. The selectivity of our method was verified by comparing diclofenac tablets to pure diclofenac. Bands used for quantification were only unique to diclofenac. Figure 2a and 2b represents the direct and first derivative of ART-FTIR spectra, which indicate the CO high absorption band at 1550–1605 cm-1. The first derivative shows a clear band without any overlapping from the other peaks. Additionally, Figure 3c and 3d represents the direct and first derivative spectra of Olfen tablet, respectively. The obtained data represented in these figures showed no interference from the excipients and additives present in the tablet formulation.\n\n(a) Spectrum of pure diclofenac sodium mixed with potassium bromide (KBr; 0.6% w/w). (b) First derivative spectrum of pure diclofenac sodium mixed with KBr (0.6% w/w).\n\n(a) Overlay spectra of different diclofenac sodium reference substance concentrations. (b) Calibration curve of diclofenac sodium showing the linear regression equation. (c) Direct Fourier transform infrared (FTIR) spectra of Olfen Tablet. (d) First derivative FTIR spectra of Olfen Tablet.\n\nThe LOD is the lowest amount of analyte at which that an analyte can be detected. While the LOQ is the lowest amount of analyte at which an analyte can be measured accurately and precisely.\n\n\n\n\n\nWhere σ is the standard deviation and S is the slope.\n\nBy applying Equation 1 and Equation 2, the obtained values of LOD and LOQ were 0.052757% and 0.159869%, respectively (Table 1). The low values for both parameters indicate the high sensitivity of this study24.\n\nThe accuracy of the method was evaluated using the calibration method. Three different brands (A, B, and C) of diclofenac tablets were analyzed. 10 tablets of each brand were crushed to a fine powder and mixed with KBr to prepare 0.5% w/w diclofenac/KBr. Mean recovery of Tablets A, B and C was calculated to be 99.8%, 101.5% and 99.4, respectively (Table 2). The obtained results are in agreement with USP and BP recovery range for DS tablets12,13.\n\nFTIR, Fourier transform infrared; DS, diclofenac sodium; API, active pharmaceutical ingredient; SD, standard deviation.\n\nThe precision of the method was assessed by repeatability and intermediate precision studies. Repeatability and robustness analysis were performed by analyzing the 0.5% w/w (mixed with KBr) peak areas (AUC) of each brand. Three readings for each sample were recorded and analyzed in one day (for the intra-day precision) and two readings were recorded on two consecutive days (for the inter-day precision). The results were very precise, as represented by the standard deviations, which ranged between 0.008343 and 0.020255, while the percent relative standard deviation (% RSD) was between 1.26095 and 3.03781.\n\n\nConclusion\n\nThe proposed first derivative ATR-FTIR spectroscopic method is considered as a green, nondestructive, low cost, fast, sensitive, accurate and precise technique for the quantitative analysis of DS in its pure and tablet dosage formulation and can be easily applied for quantitative determination and quality control.\n\n\nData availability\n\nHarvard Dataverse: Utility of ATR-FTIR Spectroscopic Method as Green Tool for Pharmaceutical Analysis of Diclofenac Sodium Tablets. https://doi.org/10.7910/DVN/6SJZ7W25.\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 are thankful to the Neopharma Pharmaceutical Co., Abu Dhabi, UAE for providing the diclofenac sodium reference standard.\n\n\nReferences\n\nScholer DW, Ku EC, Boettcher I, et al.: Pharmacology of diclofenac sodium. Am J Med. 1986; 80(4B): 34–38. PubMed Abstract | Publisher Full Text\n\nAlquadeib BT: Development and validation of a new HPLC analytical method for the determination of diclofenac in tablets. Saudi Pharm J. 2019; 27(1): 66–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArisoy GG, Dural E, Mergen G, et al.: Development and Validation of HPLC-UV Method for the Determination of Diclofenac in Human Plasma with Application to a Pharmacokinetic Study. Turk J Pharm. 2016; 13(3): 292–299. Reference Source\n\nDongala T, Palakurthi AK, Velaveni KK, et al.: Development and Validation of RP-HPLC Method for Simultaneous Determination of Diclofenac Potassium and its Process Related Impurities in Solid Oral Dosage Form. J Chromatogr Sep Tech. 2018; 9: 412. Publisher Full Text\n\nYilmaz B, Ciltas U: Determination of diclofenac in pharmaceutical preparations by voltammetry and gas chromatography methods. J Pharm Anal. 2015; 5(3): 153–160. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUzochukwu IC, Nzegbunam SO: Visible Spectrophotometric and Thermodynamic Studies of Diclofenac: Charge-Transfer Complexation with 2, 3 Dichloro-5, 6-dicyano, 1, 4-benzoquinone. Tropical J Pharm Rese. 2015; 14(3): 519–525. Publisher Full Text\n\nCastillo M, Bruzzone L: Indirect fluorometric determination of diclofenac sodium. Anal Sci. 2006; 22(3): 431–3. PubMed Abstract | Publisher Full Text\n\nParys W, Pyka-Pajak A, Dołowy M: Application of Thin-Layer Chromatography in Combination with Densitometry for the Determination of Diclofenac in Enteric Coated Tablets. Pharmaceuticals (Basel). 2019; 12(4): pii: E183. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOliveira MC, Bindewald EH, Marcolino LH Jr, et al.: Potentiometric determination of Diclofenac using an ion-selective electrode prepared from polypyrrole films. J Electroanal Chem. 2014; 732: 11–16. Publisher Full Text\n\nElbalkinya HT, Yehiab AM, Riada SM, et al.: Potentiometric diclofenac detection in wastewater using functionalized nanoparticles. Microchem J. 2019; 145: 90–95. Publisher Full Text\n\nSzostak R, Mazurek S: Quantification of active ingredients in suppositories by FT-Raman spectroscopy. Drug Test Anal. 2013; 5(2): 126–129. PubMed Abstract | Publisher Full Text\n\nUnited States Pharmacopeial Convention, 37th edn. and the national formulary, 32nd edn. United States Pharmacopeial Convention: Rockville, MD. 2013.\n\nBritish Pharmacopoeia: Her Majesty’s Stationery Office: London, UK. 2013. Reference Source\n\nNugrahani I, Dillen N: Rapid assay development of diclofenac sodium coated tablet assay using FTIR compared to HPLC method. Int J App Pharm. 2018; 10(4): 43–50. Publisher Full Text\n\nCadet F, Garrigues S, de la Guardia M: Quantitative Analysis, Infrared in the Encyclopedia of Analytical Chemistry. John Wiley & Sons, Ltd. 2012. Publisher Full Text\n\nChe Man YB, Syahariza ZA, Rohman A: Chapter 1. Fourier transform infrared (FTIR) spectroscopy: development, techniques, and application in the analyses of fats and oils. In Fourier Transform Infrared Spectroscopy. edited by Oliver J. Ress, Nova Science Publishers, New York: USA. 2010; 1–26. Reference Source\n\nMoros J, Garrigues S, de la Guardia M: Vibrational spectroscopy provides a green tool for multi-component analysis. Trends Anal Chem. 2010; 29(7): 578–591. Publisher Full Text\n\nde la Guardia M: Green analytical chemistry. Trends Anal Chem. 2010; 29(7): 577. Publisher Full Text\n\nBlanco M, Peguero A: Analysis of pharmaceuticals by NIR spectroscopy without a reference method. Trends Anal Chem. 2010; 29(10): 1127–1136. Publisher Full Text\n\nBunaciu A, Abou-Enein HY, Fleschin S: Application of Fourier Transform Infrared Spectrophotometry in Pharmaceutical Drugs Analysis. Appl Spectrosc Rev. 2010; 45: 206–219. Publisher Full Text\n\nNugrahani I, Manosa EY, Chintya L: FTIR-derivative as a green method for simultaneous content determination of caffeine, paracetamol, and acetosal in a tablet compared to HPLC. Vib Spectrosc. 2019; 104: 102941. Publisher Full Text\n\nMazurek S, Szostak R: Quantitative determination of diclofenac sodium and aminophylline in injection solutions by FT-Raman spectroscopy. J Pharm Biomed Anal. 2006; 40(5): 1235–1242. PubMed Abstract | Publisher Full Text\n\nInternational Conference on Harmonization (ICH) of technical requirements for registration of pharmaceuticals for human use, Validation of analytical procedures: Text and methodology Q2 (R1). ICH Geneva, Switzerland. 2005. Reference Source\n\nShrivastava A, Gupta VB: Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci. 2011; 2(1): 21–25. Publisher Full Text\n\nFahelelbom KM, Saleh A, Mansour R, et al.: \"Utility of ATR-FTIR Spectroscopic Method as Green Tool for Pharmaceutical Analysis of Diclofenac Sodium Tablets\". Harvard Dataverse, V1, UNF:6:5J6Gk08vUOY7ReTP51OO1g== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/6SJZ7W"
}
|
[
{
"id": "61176",
"date": "16 Mar 2020",
"name": "Yasser Fakhri Mustafa",
"expertise": [
"Reviewer Expertise Medicinal Chemistry",
"Chemistry of natural products",
"Pharmaceutical analysis",
"Organic synthesis",
"and instrumentations."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 aimed to investigate one of the green analytical chemistry tools for quantitative determination of Diclofenac sodium (DS) in tablet dosage forms by using ATR-FTIR technique. The novelty of this work arises from excluding the need for sample preparation like the using of KBr discs. I find the work interesting, informative, and worthy to be indexed. However I have some concerns/suggestions after carefully reading the report.\n2nd column in Table 2 needs consistency in the expression of concentration of DS because sample B is expressed in terms of %, while samples A&C were expressed as quantities.\n\nI would rather suggest expressing the standard deviation (SD)and relative standard deviation (RSD) in a table as follows:\nSample name\n\nSD\n\nRSD Sample A\n\n0.0083\n\n1.26% Sample B Sample C\n\nWould the authors be able to state whether this method suits coated tablet forms of DS?\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": "5324",
"date": "02 Apr 2020",
"name": "Khairi Salem",
"role": "Author Response",
"response": "Authors Response for Comments of reviewers 1 - Yasser Fakhri Mustafa and Hasan Aldewach: All data in Table 2 is represented in % w/w. Sample B data in the Table 2 was corrected according to the reviewers comments in order to be consistent with samples A and C. The standard deviations (SD) and relative standard deviations (RSD) have been summarized under precision and robustness section. This work describes the Utility of ATR-FTIR for the analysis of uncoated tablet of diclofenac sodium, as the excipients is not interfering in the analysis of active ingredient we expect that the coating material may need certain extraction process. Authors Responses for Comments of reviewer 2 Baker Jawabrah Al-Hourani:DS is known to be nonselective COX inhibitor. The inhibition of COX-1 (constitutive isoform) is assumed to be the main cause of undesirable side effects of the drug. However; the inhibition effects of the COX-2 (inducible isoform) yield the desired effects. The exceptional potency of DS among other NSAIDs was explained on the basis of the additional 5-lipoxygenase (5-LOX) indirect inhibitory effect of the drug.ReferenceFiorucci S, Meli R, Bucci M, Cirino G, Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy, Biochemical Pharmacology. 2002; 62(11):1433-8, DOI: 10.1016/S0006-2952(01)00747-XAuthor : Khairi M.S. Fahelelbom"
}
]
},
{
"id": "61173",
"date": "19 Mar 2020",
"name": "Baker Jawabrah Al-Hourani",
"expertise": [
"Reviewer Expertise Drug design and Discovery 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\nQuantitative determination of diclofenac sodium in tablet dosage was carried out by using the ATRFTIR device. This is a unique methodology and considered as a part of the green chemistry field. The work is clearly presented, reproducible, clean, and full of important information, which can be used by any third party.\nFirst comment is regarding a typo in the first paragraph in the introduction, line 5: Please change “cyclo-oxygenase” to “cyclooxygenase”.\nSecond comment is regarding the scientific content of the same paragraph, line 5: Please specify the type of the cyclooxygenase: 1/2 or both. The same thing regarding the lipoxygenase enzyme.\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": "5324",
"date": "02 Apr 2020",
"name": "Khairi Salem",
"role": "Author Response",
"response": "Authors Response for Comments of reviewers 1 - Yasser Fakhri Mustafa and Hasan Aldewach: All data in Table 2 is represented in % w/w. Sample B data in the Table 2 was corrected according to the reviewers comments in order to be consistent with samples A and C. The standard deviations (SD) and relative standard deviations (RSD) have been summarized under precision and robustness section. This work describes the Utility of ATR-FTIR for the analysis of uncoated tablet of diclofenac sodium, as the excipients is not interfering in the analysis of active ingredient we expect that the coating material may need certain extraction process. Authors Responses for Comments of reviewer 2 Baker Jawabrah Al-Hourani:DS is known to be nonselective COX inhibitor. The inhibition of COX-1 (constitutive isoform) is assumed to be the main cause of undesirable side effects of the drug. However; the inhibition effects of the COX-2 (inducible isoform) yield the desired effects. The exceptional potency of DS among other NSAIDs was explained on the basis of the additional 5-lipoxygenase (5-LOX) indirect inhibitory effect of the drug.ReferenceFiorucci S, Meli R, Bucci M, Cirino G, Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy, Biochemical Pharmacology. 2002; 62(11):1433-8, DOI: 10.1016/S0006-2952(01)00747-XAuthor : Khairi M.S. Fahelelbom"
}
]
}
] | 1
|
https://f1000research.com/articles/9-176
|
https://f1000research.com/articles/8-655/v1
|
13 May 19
|
{
"type": "Research Article",
"title": "Stress and associated risk factors among the elderly: a cross-sectional study from rural Thailand",
"authors": [
"Katekaew Seangpraw",
"Nisarat Auttama",
"Ramesh Kumar",
"Ratana Somrongthong",
"Prakasit Tonchoy",
"Pitakpong Panta",
"Katekaew Seangpraw",
"Nisarat Auttama",
"Ratana Somrongthong",
"Prakasit Tonchoy",
"Pitakpong Panta"
],
"abstract": "Background. Stress is a common mental health problem of the elderly population that affects their quality of life. The objective of this study was to determine the level of stress and associated factors among the elderly living in rural areas of Thailand. Methods. This was a cross-sectional study conducted in two sub-districts of rural Thailand and interviewed 403 elderly persons. We used simple random sampling technique from a list of registered elderly individuals and conducted face-to-face interviews using a questionnaire. The questionnaire were piloted, validated and pretested beforehand. Multiple linear regression was applied for data analysis. This study was approved by the Ethical Review Committee of the University of Phayao, Thailand. Results. The mean age of the participants was 68 and two thirds (67%) were female. Less than 43% of the participants had moderate, and one third (34%) had high levels of stress. More than half of participants had low level stress management. Stress was significantly associated with alcohol and illness with a predictive power of 3.0% [(R = 0.173, R Square = 0.030) (p<0.05)]. Conclusion. We conclude that risk factors such as alcohol and illness affect elderly population living in rural areas of Thailand to a major extent in terms of stress.",
"keywords": [
"Evaluation factors",
"stress",
"elderly rural",
"risk factors",
"association"
],
"content": "Introduction\n\nGlobally, 15% of the elderly population is suffering from mental disorders, and stress is one major mental health problem affecting a sizeable proportion (10–55%) of the elderly population1,2. The prevalence of stress and anxiety among the elderly population is gradually increasing and expected to reach double in the next one decade1. About one fifth of the world’s aging population lives in Thailand, and their number will increase by 28% in the coming ten years3.\n\nIn Thailand, recent surveys have reportedly identified increasing stress and mental health issues. Hospital based data complements this by showing increasing burden of stress and anxiety among the elderly4. Recent research has also suggested that the prevalence of stress is associated with age and the chances of getting this condition has increased in the aging population3,4.\n\nSecondary data from rural Thailand depicts a high proportion of the elderly population suffering from mental health disorders5. Research suggests that common factors affecting stress among the elderly are family relationship, financial status, social or community environment, physical health and chronic illness7–10. Nonetheless, the factors associated with stress need further exploration. Hence, we conducted this research to determine the factors affecting stress among the elderly in rural Thailand.\n\n\nMethods\n\nThis was a cross-sectional study carried out between January and April 2017 in Muang District, Phayao Province of Thailand.\n\nThe study sample size was calculated by using confidence level of 95%, the coefficient of the error = 5% and population proportion of 0.0511,12. Hence, 403 elderly people were interviewed in this study by simple random sampling method from a list of promoting hospitals1 registering elderly patients. Our tool was based on Pender’s theory of health promotion model and stress assessment13,14. We included male and female elderly persons who were above 60 years old, living in the study area for more than one year and able to communicate. However, those who were admitted with other associated diseases were excluded in this study.\n\nData collectors were trained and briefed on the study prior to conducting this survey. Face to face interviews of 40 minutes per participant were conducted. There were three parts of the questionnaire; socio-economic characteristics (age, sex, income, education, marital status etc), the stress assessment test composed of 20 items from Suangprung Stress test-20, and the stress management score (10 items may rating scale on four point Likert scale)13,14 The stress management section was adapted to the elderly community with questions pertaining to the following; “Feeling desperate in life”, “Cannot stay focused”, “Cannot sleep due to stress or overthinking”, and “Muscle pain in the back or shoulders”. The mean score was calculated from their responses; less stress (0 – 23), moderate stress (24 – 41), high stress (42 – 61) and severe stress (>62)14. The total scores were divided into three levels including low scores (0–30), moderate scores (31–39) and high scores (40–50)14. The questionnaire was piloted and pretested on 35 elderly living in outside from the study area with similar settings. Cronbach’s alpha coefficient of the questionnaire was calculated as 0.80 and content validity, a Kuder-Richardson 20 coefficient, was assessed as 0.79.\n\nData was analyzed using SPSS Statistics version 20.0. Descriptive and multiple stepwise linear regression analysis was used to investigate the potential predictors of stress among the elderly. The analysis we put in the model 1 is alcohol consumption and the model 2 is present illness like; hypertension, musculoskeletal disorders and diabetes as these were the main variables as per our objectives.. The level of significance for all statistical tests was set at p-value <0.05.\n\nAll participants were informed regarding the research objectives and procedures of the study and a written informed consent was obtained from all the participants prior to start of the study. All the information of participants was kept confidential. This study was approved by the Ethics Review Committee for research involving human research subjects at the University of Phayao Thailand (No. 2/101/59). Administrative approval was gained from the head of the hospitals before to the study began.\n\n\nResults\n\nThe mean age of study participants was 68±7, and more than half (67%) of participants were women. About half (50%) of the participants were single, having no education (62%), received monthly income less than 100 US$ (73%). Present illness was defined as having a chronic illness at time of sampling (Hypertension, musculoskeletal disease and hypertension). Around two thirds (63%) of the respondents reported a present illness; hypertension (52%), musculoskeletal disorders (29%), and diabetes mellitus (19%). About two thirds (69%) of participants lived with family members. Almost half of participants consumed alcohol (45%) and 27% smoked cigarettes (Table 1).\n\nTable 2 shows stress levels among elderly people during the last three months as calculated using the Suangprung Stress test-20 stress assessment test. Almost half of these participants experienced a moderate level of stress (43%). Around 34% experienced a high level of stress and 18% had a low level of stress.\n\nIn term of stress management during the last three months, the results showed that more than half of participants had a low level of stress management (59%), followed by moderate and high levels of stress management (33% and 8%, respectively) (Table 3).\n\nThere was statistically significant relationship between alcohol consumption and present illness with stress levels, as calculated using the Suangprung Stress test-20 stress assessment test (Table 4).\n\nModel 1 R =0.141, R2 Square = 0.020, S.E = 11.192, n =403, Model 2 R = 0.173, R2 Square = 0.030, S.E = 11.149, n =403\n\nThe stress scores is 2.95 points higher (b coefficient, Table 5) than the elderly who drink alcohol than those who did not use alcohol. This indicates use of alcohol among elderly is positively associated with their current illness, likely due to their perception that the alcohol will help with mental relaxation. In contrast, if the elderly continue consuming alcohol, the present illness will result in increased stress for the participants. (Table 5).\n\nPresent illness: no (0), yes (1); Alcohol consumption: no (0), yes (1). * = significant p-value\n\n\nDiscussion\n\nIn the present study, the majority of elderly people had moderate and high levels of stress during the last three months. This level of stress among the elderly population could negatively affect their health and well-being7,15. Other studies elsewhere have shown stress’s drafting effects, indicting that stress would directly effect mental and physical status among the elderly3,15. Our findings are consistent with a previous study15. Further according to the wear and tear theory, when the elderly population are experiencing poor physical and mental health, they would more likely to develop anxiety16,17. Chronic diseases and economic problems are the major causes of stress among the elderly. Moreover, long term stress and anxiety can also lead to depression and suicidal tendencies among the elderly9,17. Studies in South Korea and Denmark found that higher levels of perceived stress were associated with higher mortality18–20.\n\nMore than half of the elderly participants had a low level of stress management and were living with their grandchildren with their children working other areas. Hence, the elderly had to take care of all responsibilities including household, grandchildren and financial support to the family3,15. These responsibilities would tend to develop stress and anxiety among elderly. These findings are similar to a study showing emotional tension was a major contributing factor leading to mental health problems among the elderly17.\n\nIn the present study, the two main factors associated with stress among the elderly were alcohol consumption and present illness. Stressed elderly individuals usually prefer alcohol to achieve mental relaxation21. Research shows that negative feelings including stress, disappointment, hatred and unsuccessful can lead to drinking behavior21. Previous research show a strong positive correlation between stress and drinking alcohol, especially among the elderly population22. Moreover, present illness is a predictive power of stress among the elderly where current illness could influence daily life activities. Mental health problems and living in a stressful condition could impact their physical health, sleeping and quality of life23. The literature compliments our findings that chronic illnesses might affect the level of stress among elderly people24,25. A study performed on elderly people living with hypertension showed that there was a statistically significant relationship between chronic illness and stress26. Our findings are also consistent with a study on elderly people with diabetes leading to anxiety and stress, ultimately developing depression among this aging population27.\n\n\nConclusion\n\nThis study provides an understanding of current mental health situations and factors affecting stress, such as alcohol consumption and illness, of elderly people living in rural communities of Thailand. Non-communicable diseases including hypertension, diabetes, and musculoskeletal disorders are the leading factors shown to develop stress and anxiety.\n\n\nData availability\n\nOpen Science Framework: Stress and associated risk factors among the elderly: a cross sectional study from rural Thailand study, https://www.doi.org/10.17605/OSF.IO/XVKSW28\n\nThis project contains the following underlying data:\n\nData dictionary for statistic analysis plan.doc (data dictionary)\n\nUpdate Data set.xls (Participant data)\n\nOpen Science Framework: Stress and associated risk factors among the elderly: a cross sectional study from rural Thailand study, https://www.doi.org/10.17605/OSF.IO/XVKSW28\n\nThis project contains the following extended data:\n\nquestionnaire_stress.doc (study questionnaire)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nThis work was supported by the University of Phayao (Grant No.RD61058) and the Rachadapisek Sompote Fund for Postdoctoral Fellowships, Chulalongkorn University Thailand to RK.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nFootnotes\n\n1Hospitals in Thailand are operated by both the public and private sector to provide medical services for prevention, cure and rehabilitation of patients with medical and health-related conditions\n\n\nReferences\n\nWorld Health Organization: Mental health and older adults. Geneva; 2016.\n\nBabazadeh T, Sarkhoshi R, Bahadori F, et al.: Prevalence of depression, anxiety and stress disorders in elderly people residing in Khoy, Iran (2014-2015). J Anal Res Clin Med. 2016; 4(2): 122–8. Publisher Full Text\n\nSiripanich B: Situation of the elderly 2016. Foundation for Thai elderly research and development institute Bangkok, Thailand. 2017.\n\nDepartment of Mental Health: Strategic plan for the mental health during the National economic and social development plan. Ministry of Public Health Thailand, 2017.\n\nPhayao hospital Report: Situation of mental health in Phayao. Psychiatric services Phayao hospital, 2017.\n\nTantipiwatanaskul P: Caregiving to Older Persons: Happiness and Stress. Department of Mental Health: Ministry of Public Health Nonthaburi: Thailand; 2013.\n\nYaka E, Keskinoglu P, Ucku R, et al.: Prevalence and risk factors of depression among community dwelling elderly. Arch Gerontol Geriatr. 2014; 59(1): 150–54. PubMed Abstract | Publisher Full Text\n\nVasunilashorn S, Glei DA, Weinstein M, et al.: Perceived stress and mortality in a Taiwanese older adult population. Stress. 2013; 16(6): 600–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreen LW, Kreuter MW: Health promotion planning: an educational and environmental approach. 2nd ed. Palo Alto. Mayfield Publishing Co, 1991. Reference Source\n\nCochran WJ: Practical nonparametric statistics. New York: John Wiley; 1997.\n\nPhoosuwan N: The stress of elderly in a district, Sakonnakhon Province. J of Mahasarakham Uni Humanities and Social Sci. 2014; (Special journal): 164–71. Reference Source\n\nPender NJ: Health Promotion in nursing practice. 2nd ed: Connecticus: Appleton & Lange; 1996. Reference Source\n\nYodmai K, Somrongthong R, Kumar R: Impact of the Economic deficits and the Quality of Life in elderly, Rural community, Khonkean Province, Thailand. J Liaquat Uni Med Health Sci. 2018; 17(3): 180–4.\n\nKlinwichit S, Klinwichit W, Incha P, et al.: Mental health evaluation of the elderly with chronic illness in community: Saensuk Municipality Chon Buri Thailand. BJM. 2013; 2(1): 21–23. Reference Source\n\nProdbumrung J, Leeka J, Thongpan S: Health Care for the Elderly, According to the Buddhist Ethics. Buddhachinaraj Medical Journal. 2016; 33(2): 233–40. Reference Source\n\nGray R, Thapsuwan S, Thongcharoenchupong N, et al.: Caregiving to older persons: happiness and stress. Institute for Population and Social Research: Mahidol University. Thailand; 2013.\n\nKhang YH, Kim HR: Explaining socioeconomic inequality in mortality among South Koreans: an examination of multiple pathways in a nationally representative longitudinal study. Int J Epidemiol. 2005; 34(3): 630–37. PubMed Abstract | Publisher Full Text\n\nNielsen NR, Kristensen TS, Schnohr P, et al.: Perceived stress and cause-specific mortality among men and women: results from a prospective cohort study. Am J Epidemiol. 2008; 168(5): 481–91; discussion 492–6. PubMed Abstract | Publisher Full Text\n\nLuchesia BM, Souza ÉN, Gratão AC, et al.: The evaluation of perceived stress and associated factors in elderly caregivers. Arch Gerontol Geriatr. 2016; 67: 7–13. PubMed Abstract | Publisher Full Text\n\nHam LS, Hope DA: College students and problematic drinking: a review of the literature. Clin Psychol Rev. 2003; 23(5): 719–59. PubMed Abstract | Publisher Full Text\n\nSamai T, Jewpattanakul Y, Phetphansee S: The relationships between personal, socioeconomic and stress factors on alcohol drinking behavior of dwellers in Bangkoknoi District, Bangkok. J Nurs Sci. 2015; 33(1): 42–50. Reference Source\n\nHongthong D, Somrongthong R, Womgchaiya P, et al.: Factors Predictive Of Alcohol Consumption Among Elderly People In A Rural Community: A Case Study In Phayao Province Thailand. J Ayub Med Coll Abbotabad. 2016; 28(2): 237–240. PubMed Abstract\n\nPhoosuwan N: The stress of elderly in a district Sakonnakhon Province. J of the Humanities and Social Sciences. The 12th Mahasarakham University Research Conference. 2014; 165–71.\n\nJeon HS, Dunkle RE: Stress and Depression Among the Oldest-Old: A Longitudinal Analysis. Res Aging. 2009; 31(6): 661–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThongtang P, Seesawang J: Lived experience of Thai older people living with hypertension. J of The Royal Thai Army Nurses. 2014; 15(3): 288–95. Reference Source\n\nSulukananuruk C, Jaisanook W, Muktabhant B: Association of depression and stress with HbA1c level of Type 2 diabetic patients attending the diabetes clinic of Nampong Hospital, Khon Kaen Province. Srinagarind Med J. 2016; 31(1): 34–6. Reference Source\n\nEknithiset R, Somrongthong R, Kumar R: Factors Associated With Knowledge, Perception, And Practice Toward Self-Care Among Elderly Patients Suffering From Type 2 Diabetes Mellitus In Rural Thailand. J Ayub Med Coll Abbotabad. 2018; 30(1): 107–110. PubMed Abstract\n\nKumar R: Stress and associated risk factors among the elderly: a cross sectional study from rural Thailand. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/XVKSW"
}
|
[
{
"id": "48413",
"date": "29 May 2019",
"name": "Thant Zaw Lwin",
"expertise": [
"Reviewer Expertise Environmental Health and Occupational Health",
"Health Care Management",
"Biostatistics",
"Epidemiology",
"Project Evaluation",
"Quantitative and Qualitative Research",
"Health Economic",
"Maternal and Child Health",
"Drug Addictions Problems."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Abstract There was no recommendation in conclusion section, or is there a word limit for the abstract? In the keywords: there was no mention of \"evaluation factors\" in the abstract, and what do you mean by \"Elderly rural\" or \"association\"?\n\nIn Introduction 2nd paragraph; \"In Thailand, recent surveys …\" and last line in that paragraph \"Recent research …\", these two lines should shift to the conclusion section.\n\nIn Methods What about the sampling procedure?\nIn Data Collection Face to face interviews of 40 minutes and the results showed that 62% have no education, is it possible that it took too much time to get the right answers. What about their responses? This survey was pre-tested and validated outside of the study, so what are the operational definitions from this?\n\nIn Results 67% of participants were women (meaning that 33% were men). But in Table 1, alcohol consumption was 45.4%. Does this mean all the men has alcohol consumption and few of elderly women has alcohol consumption? 50% of the participants were single but in the discussion, more than half of the elderly participants had a low level of stress management and were living with their grandchildren. And in the results about two thirds (69%) of participants lived with family members. What about these two connections? In Table 4, why the sample is 402 and why not 403?\n\nIn Conclusion No recommendation or other relevant evidence for association? How about recommendations for the next study?\nIn References No. 28 reference is where citations in manuscript?\n\nOverall Conclusions Please answer my questions and correct for some facts. Its just minor corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5150",
"date": "13 Jan 2020",
"name": "Ramesh Kumar",
"role": "Author Response",
"response": "Abstract: Recommendations are included in the main paper, here we can not include due to word limit. Evaluating factors are not included in our objectives, elderly rural are the key words.Introduction: Recent does not mean about this research, we are talking about other recent research to triangulate with our work. Hence it is possible to move in conclusion section.Methods: Simple random sampling method was adopted. Face to face interview was guided by the data collectors and findings were included in the result section.The questionnaire was piloted and pretested on 35 elderly living in outside from the study area with similar settings. Cronbach’s alpha coefficient of the questionnaire was calculated as 0.80 and content validity, a Kuder-Richardson 20 coefficient, was assessed as 0.79.Results: Table 1 findings are truly representing our findings. We have mentioned 50% like nearly half in the results. Typo error will be corrected by 403 in table 4.Conclusion: Recommendations are not included as per the Journal's criteria.References: 28 will be added with 27 in the list"
}
]
},
{
"id": "53600",
"date": "24 Sep 2019",
"name": "kraichat Tantrakarnapa",
"expertise": [
"Reviewer Expertise Public Health",
"Environmental Health",
"Tropical medicine. Climate change and health impacts."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Title:\nIt seems to be clear. However, it was conducted only in sub-districts in one province of Thailand. The title is a big one; how can this study represent the rural area of Thailand? There were 7,435 sub-districts in 2018. Only two sub-districts were selected as the representative of the rural area.\n\nIntroduction:\n“Hence, we conducted this research to determine the factors affecting stress among the elderly in rural Thailand.” It was indicated in the manuscript. The title and the significance of this study focus on the overview of Thailand, whereas the study areas examined were only 2 sub-districts and Muang District (city). Can they be representatives for rural areas? As indicated in Research methodology.\n\nMethods:\n“The study sample size was calculated by using a confidence level of 95%, the coefficient of the error = 5% and the population proportion of 0.05.” Authors got 403 sample size. What is the formula for sample size calculation?\n\n“Hence, 403 elderly people were interviewed in this study by simple random sampling method from a list of promoting hospitals registering elderly patients”. Do the researchers collect only elderly patients? Can they be the representatives for elderly people there?\n\nAre the subjects Thai nationality? In this province, there might be some migrants who lived in this province for more than one year and they can communicate in Thai?\n\n“Face to face interviews of 40 minutes per participant were conducted”. Is it a digit for interviewing time? Time is used for controlling the interview.\n\nAuthors indicated that the tools were tested prior to usage. Is IOC used for testing the content validity? For example, “Cannot sleep due to stress or overthinking”, the question was asked directly to the point of stress, is it biased?\n\nCronbach’s alpha coefficient of the questionnaire was calculated as 0.80. It might be the total score of a questionnaire?\n\n“Kuder-Richardson 20 coefficient, was assessed as 0.79.” Please clarify the meaning of this statement.\n\nData were analyzed using SPSS Statistics version 20.0. Is this a licensed software?\n\nModel 2 is a present illness like; hypertension, musculoskeletal disorders and diabetes. Does this variable count for “existing illness” and “no existing illness” or consider the number of existed diseases for a regression analysis?\n\nAdministrative approval was gained from the head of the hospitals before the study began. Does this study focus on the elderly in the hospital?\n\nResults:\nHaving no education, could the authors use another word replacing “no education”?\n\nIncome (per month US$).\n\nThe researchers did it during 2017, at that time the Thai Baht value is different from the current situation (please specify the exchange rate).\n\nSmoking and alcohol drinking status, the authors classified only 2 categories “Yes” and “No” at the interviewing time. The experience of consumption or smoking were not considered. Some just quit or stop smoking or drinking, are they influencing the stress?\n\nIn term of stress management as indicated in the manuscript. Please describe more details of this issue. What are they? For example; low level of management, what does it mean?\n\n“This indicates the use of alcohol among the elderly is positively associated with their current illness, likely due to their perception that the alcohol will help with mental relaxation.” Is there any scientific information supporting this statement?\n\nFor the results indicated in Table 5, are there any perception variables used in the model? Because of drinking, are they facing the stress or they have stress then they decided to release it by drinking?\n\nConclusion:\nIt is not clear for the conclusion and recommendation is not available there.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5151",
"date": "13 Jan 2020",
"name": "Ramesh Kumar",
"role": "Author Response",
"response": "Title and introduction: we have selected two sub-rural districts in the study randomly that was truly representative of this rural population.Methods:1. We used the confidence level of 95%, the coefficient of the error = 5% and population proportion of 0.05. 2. Yes, only elderly population were included in this study.3. Yes only Thai national were included.4. Yes we agree.5. No, we have asked only those questions were included in the tool.6. Yes this was Cronbach’s alpha coefficient of the questionnaire was calculated as 0.80.7. Kuder-Richardson 20 coefficient, was assessed as 0.79 was used to check validity of the questionnaire.8. Yes that was licence software.9. Only existing illness.10. Administrative approval from hospital was sought to include elderly population in the study from the registered record available with them.Results: 1. No.2. Yes converted thai bath in the US$3.Conversion rate was taken at that time.4. Included those who were actively smoking or not5. We have categorised the stress level and measured accordingly.6. This information supported in the discussion part.7. Yes.Conclusion: We have achieved our objectives and concluded accordingly and did not included the recommendation as per the Journal's criteria."
}
]
}
] | 1
|
https://f1000research.com/articles/8-655
|
https://f1000research.com/articles/9-225/v1
|
01 Apr 20
|
{
"type": "Opinion Article",
"title": "The consequence of COVID-19 on the global supply of medical products: Why Indian generics matter for the world?",
"authors": [
"Philippe J Guerin",
"Sauman Singh-Phulgenda",
"Nathalie Strub-Wourgaft",
"Sauman Singh-Phulgenda",
"Nathalie Strub-Wourgaft"
],
"abstract": "While the world is facing the urgency of the COVID-19 pandemic, policymakers must plan for the direct response to the outbreak while minimising its collateral impact. Maintaining the supply chain of pharmaceutical products is not only paramount to cover the immediate medical response but will be fundamental to reducing disruption of the healthcare delivery system, which requires constant medicines, diagnostic tools and vaccines for smooth functioning. In this equation, the role of the Indian pharmaceutical industry will not only be critical to meet the domestic need of over 1.3 billion inhabitants but will equally be important for the rest of the world, including wealthy economies. Preventing a significant disruption of the Indian pharmaceutical supply chain during the outbreak and preparing it for large scale production for COVID-19 therapeutic or preventive medical products will not only help India but will assist the global response to this outbreak.",
"keywords": [
"generic drugs",
"COVID-19",
"India",
"active pharmaceutical ingredients",
"manufacturing industry",
"regulatory agency",
"medicine access",
"pharmaceutical industry"
],
"content": "\n\nThe COVID-19 pandemic has emerged as an unprecedented global health crisis. Although the extent of the ramifications is still to be established, it is evident that it will have a major impact on global trade in the immediate as well as distant future1. The supply chain of global pharmaceuticals is likely to be interrupted, and the impact on global access to medicine, particularly in low-and middle-income countries (LMICs), will have dramatic consequences.\n\nIn 2018–19, India exported nearly $19 billion worth of pharmaceuticals to more than 200 countries, from the highly regulated markets of North America and Europe to countries with limited pharmaceutical industry capacity such as most of sub-Saharan Africa (SSA)2. The Indian Department of Pharmaceuticals reports that formulations and biologicals account for 77% of the total Indian exports and Indian firms provide 20% of the global supply of generics3.\n\nIndian firms thus meet 40% of the generic demand in the US and a quarter of that of Europe4,5. India accounts for 12% of all manufacturing sites catering to the US market, and 50 Indian firms have a combined abbreviated new drug application (ANDA) market authorization for over 5000 medical products. Similarly, India has 622 sites approved by the European Union and nearly 1700 products with market authorization from the UK Medicines Healthcare Regulatory Agency6. Overall, SSA imports nearly 70% of its pharmaceutical needs7, and India was also the single largest supplier of medicines to Africa in 2018, accounting for a fifth of its pharmaceutical imports8.\n\nIndian firms import almost 70% of their bulk drugs from China, where the production of active pharmaceutical ingredients (APIs) and supply chain logistics have taken a big hit due to the novel coronavirus outbreak9. At the same time, the Indian government has restricted the export of 26 bulk drugs and their formulations10, including several products part of the World Health Organization (WHO) essential medicine list, e.g. antibiotics such as clindamycin, erythromycin, chloramphenicol, one antiretroviral acyclovir, which in total account for 10% of all Indian exports according to Reuters11. India’s production capacity, as well as its export potential, are in other words, already impacting pharmaceutical access in a significant manner.\n\nFurthermore, Indian manufacturers represent 67% (379) of the 563 WHO prequalified pharmaceutical products for a range of conditions such as diarrhoea (1), hepatitis (13), HIV/AIDS (197), influenza (10), malaria (41), neglected tropical diseases (3), reproductive health (21), and tuberculosis (93). A total of 130 of these products are dependent on APIs sourced from China. Besides, 15 Chinese firms are also manufacturing 42 WHO prequalified products12.\n\nUndoubtedly, the most vulnerable to the shock of the destabilization of the Indian industry will be the institutional markets for medicines in LMICs. Indian firms indeed account for over 90% of the antiretroviral (ARV) procurement in LMICs funded through donor procurement13. Two-thirds of the medicines used by important global health players like Médecins Sans Frontières (MSF) to treat HIV, tuberculosis and malaria are generics sourced from India, as well as are treatments for some neglected tropical diseases14. Likewise, 70% of the supply of pentavalent vaccines to UNICEF is dependent on the supply by Indian firms15, while a single eligible Indian firm ‘Serum Institute of India’ supplies the measles vaccine for the Gavi program16.\n\nIn the context of the COVID-19 pandemic, global reliance on Indian generics is likely to become a complex international challenge. There are no reliable substitutes for API supplies, nor production capacity available, and more importantly, any country potentially capable of establishing manufacture is likely to focus on national needs and not on export nor development aid.\n\nMitigation and control of the outbreak of COVID-19 in India are thus of paramount importance not only to India but to the world. Its capacity to import raw materials, manufacture and export medicines will not only determine how the majority of LMICs will be able to respond to the outbreak but will also affect high-income countries. As a major component of the Indian economy, the state of its pharmaceutical industry will also determine the impact of the pandemic on one-fifth of the world’s population. Historically, India has shown commitment at the highest level to ensure the health of millions around the world with a dynamic and resilient pharmaceutical industry. Exceptional measures should be taken in order to support and maintain the operationality of the production plants.\n\nGovernments and international organizations who depend on India for their supplies should look beyond their individual demands and support the Indian pharmaceutical supply chain. There is a need to look at contingency plans to assure access to APIs and medicines globally. It is expected that in a few months, diagnostic tools, medicines and vaccines will be approved by medicine regulatory agencies to diagnose, treat and prevent COVID-19 infections. Production at a large scale of those pharmaceutical goods will require the full support of the entire global pharmaceutical industry. Considering the production capability of Indian firms, their engagement will be critical for the rest of the world as well as India to return to some sort of normality post pandemic.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nEvans D, Over M: The Economic Impact of COVID-19 in Low- and Middle-Income Countries. Cent Glob Dev. 2020; (accessed March 15, 2020). Reference Source\n\nDepartment of Commerce: Export Products (Pharmaceuticals). (accessed March 12, 2020). Reference Source\n\nDepartment of Pharmaceuticals: Annual Report 2019-20. New Delhi, 2020. Reference Source\n\nIndia Brand Equity Foundation: Pharmaceuticals. New Delhi, 2019. Reference Source\n\nDasgupta N, Burger L: Europe ‘panicking’ over India’s pharmaceutical export curbs: industry group - Reuters. Reuters. 2020; (accessed March 14, 2020). Reference Source\n\nPharmexcil: 15th Annual Report 2018-2019. New Delhi, 2019. Reference Source\n\nPheage T: Dying from lack of medicines. Africa Renew. 2017; (accessed March 21, 2020). Reference Source\n\nTrade Map: Trade statistics for international business development. (accessed March 12, 2020). Reference Source\n\nChatterjee P: Indian pharma threatened by COVID-19 shutdowns in China. Lancet. 2020; 395(10225): 675. PubMed Abstract | Publisher Full Text\n\nPress Information Bureau: Government amends Export Policy of APIs and formulations made from these APIs. 2020; (accessed March 12, 2020). Reference Source\n\nThomas C, Dasgupta N: Global supplier India curbs drug exports as coronavirus fears grow. Reuters. 2020; (accessed March 12, 2020). Reference Source\n\nWorld Health Organizaton: Medicines/Finished Pharmaceutical Products. WHO - Prequalification Med. Program. 2020; (accessed March 14, 2020). Reference Source\n\nSagaon-Teyssier L, Singh S, Dongmo-Nguimfack B, et al.: Affordability of adult HIV/AIDS treatment in developing countries: modelling price determinants for a better insight of the market functioning. J Int AIDS Soc. 2016; 19(1): 20619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMédecins Sans Frontières: At EU-India Summit, India must defend its ‘pharmacy of the developing world’. Press Release. 2016; (accessed March 12, 2020). Reference Source\n\nUnicef Supply Division: Pentavalent vaccine (DTwP- HepB-Hib): Market & Supply Update. 2017. Reference Source\n\nGavi: Supply and Procurement Roadmap: Measles-Rubella (MR). 2017. Reference Source"
}
|
[
{
"id": "61902",
"date": "07 Apr 2020",
"name": "Agnès Saint-Raymond",
"expertise": [
"Reviewer Expertise International regulatory Affairs. Clinical trials",
"Paediatrics and Orphan drugs"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe opinion article is well written and extremely documented, and the statements well supported by figures and data. It aims at giving a voice to Low-and-Middle Income Countries (LMICs) which are both affected by COVID-19, but also are going to be hit hard by the supply chain disruption in India. India and the rest of the world rely heavily on drugs, mostly generics, and vaccines manufactured in India. India is the main manufacturer of vaccines for UNICEF and of WHO prequalified medicines, i.e. those needed to respond to the major disease burden of LMICs. India itself depends on China, which produces about 80% of its APIs and has already been severely affected by COVID-19 as of December 2019. The pandemic is now affecting India. India is reacting like many countries by protecting its own and putting an export ban on many essential medicines. The disruption of the supply chain will impact LMICs, as they have no or very limited capacity and capability to manufacture and certainly to compensate swiftly. They cannot compete to procure drugs if prices are increased. There is a need for global cooperation and rapid action to maintain the supply chain from India towards LMICs.\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes the article provides extensive evidence of what is the supply chain and its interdependencies. It clearly identifies the high level of dependence on India (and China) by LMICs.\n\nAre all factual statements correct and adequately supported by citations?\nThe authors highlight the complexity of the relation between China and India and the high dependency on Indian manufacturers for essential medicines for LMICs. All references are recent (2016-2020).\nAre arguments sufficiently supported by evidence from the published literature? In order to mitigate the risk of supply disruption, it would be good to recall some of the factors contributing to the world’s dependency on India and China in LMICs as well as HMICs (for example 80% of EU medicines are manufactured in either India or China). Such dependency has increased dramatically over the years, due to both countries’ investments in pharmaceutical business and competitiveness, resulting in much lower costs of medicines. Low cost was always hailed as a success, as it allows accessible and affordable medicines in LMICs. What was overlooked was the dependency and its potential consequences in case of severe disruptions of the whole of India, China or both, which could have been predicted from any viral pandemic. Causes are not the focus of this opinion, but this vulnerability will remain. The reasons for low costs would require a specific analysis (e.g. competitiveness, patent laws, state subsidies, economy of scale, social and environmental aspects) and would justify a separate article.\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? The conclusion is very clear and in line with the arguments. It is however quite general, stopping short of making proposals on how to mitigate the risks of supply chain disruption for LMICs. For example, to ensure equitable supply of limited production, which neutral body or international organisation(s), should or could take the lead? When a large proportion of the world including India is in lock-down, the countries’ economies are hard hit or even crashing, without a vaccine immediately available to limit the spread, it will take more than goodwill to reverse the national priority or protection approaches (in India and elsewhere), and to ensure international collaboration towards equitable supply. Some ideas should be added to the conclusion.\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": "63976",
"date": "09 Jun 2020",
"name": "Narcyz Ghinea",
"expertise": [
"Reviewer Expertise Pharmaceutical policy."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 aims to highlight the importance of the Indian pharmaceutical industry for the global pharmaceutical supply chain and the important role it will play in developing therapeutic goods for COVID-19. I offer the following suggestions for further refinement of the paper.\nComment 1: The authors state that India supplies 20% of the worlds global supply of generics. They then state that India provides 40% of supply in the US and 25% in Europe, 70% in sub-Sahara Africa, and 20% in Africa. From these figures it would appear that India provides more than 20% of the global supply. Could these figures be reconciled?\nComment 2: While the emphasis in this article is about the critical role the Indian pharma industry plays in the global supply of medicines, one key lesson from the COVID-19 crisis is that there is a strong need to diversify supply chains, rather than falling back into pre-existing \"critical\" dependencies. This applies both to supply of APIs and manufacturing.\n\nComment 3: The authors mention 26 bulk drugs account for 10% of all Indian exports. However it is actually 10% of all pharmaceutical exports.\n\nComment 4: It makes more sense to say Indian pharmaceutical companies \"manufacture\" or \"export\" 67% of WHO prequalified products, rather than \"represent 67%\" of these products.\nComment 5: In the last paragraph the authors state \"Governments and international organizations who depend on India for their supplies should look beyond their individual demands and support the Indian pharmaceutical supply chain\". I am not sure what demands are being referred to - could the authors elaborate?\nComment 6: I am not sure whether the fact that India accounts for 20% of the current global supply of generics translates into their engagement being \"critical for the rest of the world['s]\" future response to COVID-19 as the authors conclude. Other countries clearly have capacity to produce generics (and are currently producing 80% worth), so how the race to fulfil demand plays out is yet to be seen.\n\nComment 7: Given the clear reliance by what the authors call \"institutional markets\" on the Indian industry, perhaps the focus of the paper should be on these markets? This would be more convincing to me. For instance, in the conclusion the authors could note that since a majority of donor procured medicines used to treat HIV, tuberculosis, malaria and other tropical diseases in LMICs countries are supplied by India, it makes sense that India will play a critical role in supporting any COVID-19 response in these countries.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "63974",
"date": "09 Jun 2020",
"name": "Yves-Marie Rault Chodankar",
"expertise": [
"Reviewer Expertise global value chains",
"pharmaceutical market",
"India"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 draw on secondary data from international institutions and recent research in economics (2016-2020) to highlight the role of India as a major global supplier of pharmaceuticals, particularly to low-and middle-income countries. Writing amid the Covid-19 pandemic, they argue that the disruption of pharmaceutical supply chains due to increases in demand and restrictions on the trade of certain products (e.g. export ban on 26 bulk drugs and formulations manufactured in India) is a major threat to global health and plead for adequate political intervention for the maintenance of a smooth international trade. The article is well documented, written clearly and concisely, and borrows on diversified and reliable data sources to provide figures that are very relevant and significative.\nThis article is essential in that it evaluates the importance of India as a global supplier of pharmaceuticals at a time when rigorous data are needed to adequately inform emergency public policies which are too often shaped around political and nationalistic considerations, without looking at the scientific evidence. However, the argument would have likely been stronger if the authors had researched into the products required to fight Covid-19 (e.g. 6-digits level trade data from the UN Comtrade based on the Harmonized System of the World Customs Organization). Although we would have appreciated more attention to the specificities of the pharmaceutical products produced in India, the authors do stress that India’s role is more prevalent in the manufacturing of generic formulations and highlight their dependence on Chinese bulk drugs. In that regard, it would also have been precious to rapidly compare the role of other countries in the global supply of pharmaceuticals like China or Brazil to highlight India's particularities more sharply.\nAlthough this is not specified in the article, we assume that the main recommendation of the authors on the need to ensure the continuity of international supply chains is meant on a short-term basis, to deal with the immediate aftermath Covid-19 pandemic. We, however, miss here a more specific set of recommendations to achieve this objective, i.e. what is meant by the “full support of the entire global pharmaceutical industry”. It would also have been interesting to discuss whether the current pharmaceutical supply systems, in which countries and firms are increasingly made interdependent, should be modified to ensure more national self-reliance on the long run (e.g. through higher custom duties or incentives to relocate manufacturing units).\n\nBut all-in-all, this article accurately emphasizes the important role of India in global access to health and can be authoritative as a reference for future research and policies on pharmaceutical supply chains.\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": "63973",
"date": "10 Jun 2020",
"name": "Rory Horner",
"expertise": [
"Reviewer Expertise Pharmaceutical value chains",
"India",
"Africa",
"industrial and trade policy",
"globalisation."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article highlights the major role of India in supplying medicines to the world, relatively well-known to those who specialised in this area of research, but less to the wider world. It is important to have this message at the time of a global pandemic.\n\nThe data on the significance of India’s pharmaceutical imports in Africa – that India contributes one-fifth of its pharmaceutical imports – to some extent underestimates its significance because it is a value number rather than a volume number. More broadly, because of its role supplying generic medicines, which are usually lower-cost, India’s significance is most prominent in volume terms.\n\nThere are two slightly different dynamics that need to be considered vis-à-vis COVID-19 and India’s pharmaceutical industry: 1) the wider impact on the general supply of medical products from India (and maintaining that during the lockdown); 2) India’s role as a supplier of potential COVID-19 treatments (e.g. drugs) or of vaccines to prevent COVID-19 infection.\n\nThe focus of this piece is more on the former, partly reflecting the time the article was written and the major concerns during the initial weeks of the spread of the COVID-19 crisis and India’s lockdown. Most Indian pharmaceutical firms have managed to maintain production during lockdown, albeit at reduced levels. Many of the initial export restrictions placed in India, although subject to considerable media attention beyond India especially, were relatively short-lived. Companies with pre-existing advanced market commitments were quickly issued with exemptions from the export bans.\n\nThe second aspect – related specifically to COVID-19 treatments - is also a crucial aspect of why “Indian generics matter for the world”1 but is only briefly touched on in the latter part of this article. In the just over two months now since this article was originally published, and as Indian companies have managed to maintain some level of production during lockdown, this latter aspect has become especially significant.\n\nConsiderable contestation and international attention has surrounded India’s role in supplying hydroxychloroquine, Remdesevir and a potential vaccine. This aspect is especially important in the need for continued attention to India’s pharmaceutical industry during the pandemic, and is why key stakeholders from elsewhere may need to collaborate with India. For example, Serum Institute is already involved in manufacturing the vaccine candidate being developed at Oxford University which AstraZeneca has a license for, while Gilead has issued licenses to a number of companies in India to produce Remdesevir. A tension between globalisation and nationalism is thus likely to continue to be a key aspect of the Covid-19 response.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-225
|
https://f1000research.com/articles/9-221/v1
|
31 Mar 20
|
{
"type": "Study Protocol",
"title": "Serotypic distribution and antimicrobial resistance of Streptococcus pneumoniae in Chinese children under 5 years after the introduction of the 13-valent conjugate pneumococcal vaccine: protocol for a scoping review",
"authors": [
"Xu Han",
"Niurka Molina Águila",
"Haiyang Yu",
"C. Nivaldo Linares Pérez",
"C. María Eugenia Toledo Romaní",
"Xu Han",
"Niurka Molina Águila",
"Haiyang Yu",
"C. Nivaldo Linares Pérez"
],
"abstract": "The World Health Organization (WHO) recommends that pneumococcal conjugate vaccines (PCVs) be included in immunization programs worldwide. In China, the 7-valent pneumococcal conjugate vaccine (PREVNAR 7®) was authorized in 2008 but was not included in the national immunization programs. In 2016, PREVNAR 13®, a 13-valent pneumococcal conjugate vaccine (PCV13), was licensed for optional use in China. We will conduct a scoping review of the distribution of serotypes and antimicrobial resistance of Streptococcus pneumoniae in children aged under 5 years in China since the introduction of PCV13. We will obtain data from PubMed, the China National Knowledge Infrastructure (CNKI), and Wanfang Med Online. We will also review epidemiological data from WHO and the China Antimicrobial Surveillance Network (CHINET). Our analysis will include the condition of interest, the intervention, and the geographical region. All types of studies will be eligible for inclusion in the study database if they meet the inclusion criteria. This scoping review is intended to outline how S. pneumoniae serotypes are distributed, and it will map their antimicrobial resistance in children aged under 5 years in China. The results of this study will provide useful information on the impact of PCV13 in China.",
"keywords": [
"Streptococcus pneumoniae",
"serotype",
"antimicrobial resistance",
"13-valent pneumococcal conjugate vaccine",
"China"
],
"content": "Introduction\n\nPneumonia accounts for 15% of all deaths of children under 5 years old, killing 808,694 children in 2017; Streptococcus pneumoniae is the most common cause of bacterial pneumonia in children1. Although the widespread use of PCVs has decreased the burden of invasive pneumococcal disease (IPD)2, S. pneumoniae is still a substantial issue because immunization causes serotype changes, the available PCVs are costly, and widespread antibiotic use during the last decades has resulted in drug-resistant strains of S. pneumoniae3. The introduction of pneumococcal vaccines has decreased the incidence of drug-resistant strains in some areas4. However, because of the widespread abuse of antibiotics and limited use of vaccines in many Asian countries, antibiotic resistance remains a very serious problem in Asia.\n\nIn China, pneumococcal disease is a major cause of morbidity and mortality in children5,6. Although PCV7 was licensed in China in 2008, it was not included in the national immunization program. The coverage rate of this vaccine is low; only 10% of Chinese children received PCV7 in 2016.7 An analytical decision model indicated that the use of PCV7 could have prevented more than 16.2 million cases of pneumococcal disease and 709,411 deaths in China during the 10 years after the vaccine’s introduction8. Similarly, although PCV13 was licensed for optional use in November 2016, it is not widely used because of low awareness and the high price of the vaccine.\n\nChina still lacks a systematic surveillance program for pneumococcal diseases. To date, studies analyzing serotypes and antimicrobial resistance of S. pneumoniae have not specified the date of introduction or the type of PCV. This study may provide useful information on the impact of PCV13 in China.\n\n\nObjectives\n\nIn this study, we aim to investigate how S. pneumoniae serotypes are distributed and to map antimicrobial resistance in children under age 5 years in China since the introduction of PCV13. We formulated our main review question using the SPICE framework (setting, perspective, intervention, comparison, evaluation). The framework and key elements are summarized in Table 1.\n\n\nMethods\n\nAll types of studies are eligible for inclusion in our review if they meet all 5 of the following criteria: (1) Analyze serotype distribution or antimicrobial resistance to S. pneumoniae in China; (2) contain information on the impact of PCV13 in China; (3) include only subjects less than 5 years of age; (4) be written in English or Chinese; and (5) be published between November 2016 and February 2020.\n\nTwo investigators will independently evaluate the titles and abstracts of all articles according to the inclusion criteria. Any disagreement will be resolved through discussion among team members, and if there is difficulty related to the selection of studies, the criteria will be refined.\n\nThe study will begin in March 2020 and will include searches of PubMed, the China National Knowledge Infrastructure (CNKI), Wanfang Med Online, the WHO database, and the China Antimicrobial surveillance Network (CHINET).\n\nPubMed is the largest database of medical information in the world. Created in 1946 by the National Library of Medicine, it includes the MEDLINE database, which contains information from more than 24 million articles published in 5,600 journals in 30 languages.\n\nCNKI, as the largest, most-used online academic library in China, is a key national information construction project under the lead of Tsinghua University. It is the most comprehensive repository of knowledge in China, with more than 16 million website visits each day. In 2019, the website recorded 2.3 billion full-text downloads.\n\nWanfang Med Online aims to become a type of bridge that connects medical professionals and researchers in China with medical professionals and researchers around the world. At present, the English version includes about 200 selected Chinese medical journals including the top 115 medical journals published by the Chinese Medical Association, 1 Science Citation Index (SCI) journal, 7 Science Citation Index Expanded (SCIE) journals, and 60 Medline journals.\n\nThe search strategy will be based on a combination of three elements: the condition of interest, the specified intervention, and the geographical region (Figure 1). The use of \"OR\" and \"AND\" to combine key words is shown in Table 2.\n\nData management. Retrieved records will be automatically or manually exported into EndNote.\n\nSelection process. The search and selection process will be documented in a PRISMA flowchart9. Screening and selection will be done in duplicate by two members of the review team. Any discrepancies discovered during screening of full-text papers will be discussed with the principal investigator.\n\nData collection process. Two investigators will independently extract data from the studies that meet the inclusion criteria. Any disagreement will be resolved through discussion, and if they do not agree, the principal investigator will arbitrate. If data are missing or not sufficiently described in the studies, we will contact the corresponding author to obtain the missing information.\n\nData items. The information technology department will develop two tables for organizing the data: a table that itemizes serotype, and a table that lists antimicrobial resistance to pneumococci. The tables will contain the elements listed in Table 3.\n\nOur review will focus on the scope of the available literature about the serotype distribution of and antibiotic resistance to S. pneumoniae, and the potential impact of PCV13 in China. We do not foresee outcome prioritization, but the approach will be descriptive.\n\nThe risk of bias will be determined independently by two study team members. For nonrandomized studies, we will use the ROBINS-I tool10, and for randomized trials, or interventions, we plan to use Cochrane Collaboration's risk-of-bias assessment tool11.\n\nA PRISMA flow chart and the methodological process will be described in detail for transparency. It will indicate the evidence identified and whether it was selected for inclusion in the review. If a study is excluded, the reasons will be provided.\n\nThe data will be summarized in the form of a figure, diagram, or table. A narrative summary of the findings will also be included. The strategy for synthesizing the data involves the use of analytical methods and will perform a descriptive synthesis of the data to map the distribution of serotypes and antimicrobial resistance of pneumococci in children under 5 years of age in China after the introduction of PCV13.\n\nThe review team is presented in Table 4.\n\nThis review is a part of ongoing research in China on the interchangeability of PCV13 with the Cuban vaccine PCV7-TT. When we complete this scoping review, we will consider conducting a systematic review, and we intend to publish the results in an international journal. While preparing the present protocol, we performed preliminary searches to get an idea of the size of the available literature.\n\nBecause the scoping review methodology consists of reviewing and synthesizing the data already published, this part of the study is not subject to ethical approval.\n\nThis systematic scoping review will be the first to map antimicrobial resistance to and distribution of pneumococcal serotypes in children less than 5 years of age in China after the introduction of PCV13.\n\nIt will present an overview of all studies on serotype distribution and antimicrobial resistance, and the analysis will clarify the characteristics and possible differences between the provinces of China.\n\nA search of three large databases will enable us to create a comprehensive map of antimicrobial resistance to and distribution of pneumococcal serotypes in China.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nPneumonia. WHO, 2019. 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\nBalsells E, Guillot L, Nair H, et al.: Serotype distribution of Streptococcus pneumoniae causing invasive disease in children in the post-PCV era: A systematic review and meta-analysis. PLoS One. 2017; 12(5): e0177113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong JH: Advances in pneumococcal antibiotic resistance. Expert Rev Respir Med. 2013; 7(5): 491–8. PubMed Abstract | Publisher Full Text\n\nChinese Maternal and Child Health Development Report:Chinese Journal of Women and Children Health. 2019; 10(1). Reference Source\n\nHuang G, Shi Q, Chen H, et al.: Bacteria detection in sputum and analysis of bacterial resistance in childhood pneumonia. Journal of HeBeijing Medical University. 2016; 37(1): 40–3. Reference Source\n\nLiu S, Hu HL, Yang YH, et al.: A systematic review about Streptococcus Pneumoniae serotype distribution in children in mainland of China before the PCV13 was licensed. Expert Rev Vaccines. 2017; 16(10): 997–1006. PubMed Abstract | Publisher Full Text\n\nHu S, Shi Q, Chen CI, et al.: Estimated public health impact of nationwide vaccination of infants with 7-valent pneumococcal conjugate vaccine (PCV7) in China. Int J Infect Dis. 2014; 26: 116–22. PubMed Abstract | Publisher Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSterne JA, Hernán MA, Reeves BC, et al.: ROBINS-I: a tool for assessing risk of bias in non-randomised studies of interventions. BMJ. 2016; 355: i4919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShuster JJ: Review: Cochrane handbook for systematic reviews for interventions, Version 5.1.0, published 3/2011. Julian P.T. Higgins and Sally Green, Editors. Res Synth Methods. 2011; 2(2): 126–30. Publisher Full Text"
}
|
[
{
"id": "62038",
"date": "09 Apr 2020",
"name": "Guanlan Zhao",
"expertise": [
"Reviewer Expertise Epidemiology",
"Occupational Health",
"Cohort Studies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe in details the highlights of this study protocol. Pneumococcal disease is one of the main causes of morbidity and mortality in children under 5 years old. In 2018, the number of newborns in China exceeded 15 million, and there is a huge clinical demand for the vaccine. In China, there are two types of vaccines. The first type of vaccine refers to the vaccine provided by the government to public free of charge, and the public shall be vaccinated in accordance with the government's regulations, mainly with the routine immunization vaccine for children and the second type of vaccine refers to the vaccine that is provided by the public at their own expense and voluntarily, such as Streptococcus pneumoniae vaccine. Due to the huge burden of disease and the acknowledgement of protecting the children’s’ health about pneumococcal disease, it also shows the importance and feasibility of this study. I think it be useful to create a comprehensive map of this topic in China.\n\nAbout information sources, the author chooses the China National Knowledge Infrastructure (CNKI), Wanfang Med Online, there are two important Chinese databases. I believe that the integration of these database together with Pubmed will make the future scoping review more complete and perfect.\n\nPossible recommendation of future scoping review: In recent years, China's economy has developed rapidly, but the gap of economic level still exists in different regions of China, and the distribution of medical resources will also be affected. Is there known to be differences in education levels or income levels in selected articles? It might be good to discuss what possible other socioeconomic differences might exist in geographical regions that would account for differences in the vaccination rate in the scoping review.\n\nThe authors describe the objective and material and methods of study clearly.\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": "5422",
"date": "16 Apr 2020",
"name": "Xu Han",
"role": "Author Response",
"response": "Thank you for your comments and suggestions!We agree with your comments, it is very interesting that you mentioned about these factors affecting immunization coverage level in China, especially the impact caused by the socio-economic differences between the geographic regions."
}
]
},
{
"id": "66175",
"date": "30 Jul 2020",
"name": "Eric Bonnet",
"expertise": [
"Reviewer Expertise bioinformatics",
"biology",
"immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors describe a protocol for a scoping review of the distribution of serotypes and antimicrobial resistance of Streptococcus pneumoniae in children aged under 5 years in China since the introduction of the 13-valent conjugate pneumococcal vaccine (PCV) in China in 2016.\n\nPneumonia accounts for quite a large number of deaths for children under 5 years old worldwide and Streptococcus pneumoniae is the most common cause of the disease. In China, approximately 30,000 children die of pneumonia each year. The introduction of PCV has quite dramatically reduced the impact of the disease in many areas of the world. However, infections by S. pneumoniae are still a problem because of the many serotypes for this bacteria and also because immunization triggers serotype changes. In China, PCVs were introduced quite recently (2008), with only 10% of the children that received the vaccine. Furthermore, there is no systematic surveillance program for pneumococcal diseases. Therefore, the main goal of the protocol described in this study is very timely and will be very useful for researchers and epidemiologists.\n\nThe article is well written, concise, and with enough precision. The authors focus on the PCV 13-valent which was introduced in China in 2016, and they intend to analyze all the relevant literature published on the subject till February 2020. The criteria defined for the selection of the articles are well described and relevant. The authors intend to use three main databases to conduct their survey, namely PubMed, the China National Knowledge Infrastructure (CNKI) and Wanfang Med Online. Review epidemiological data will also be included from the World Health Organization and the China Antimicrobial Surveillance Network (CHINET).\n\nI only have a few remarks that could help to improve further the quality of the manuscript:\n\nIn the methodology section, the authors mention a potential “risk of bias”, but do not elaborate much on that topic. I would suggest explaining what this risk is about and if possible give one or two concrete examples.\n\nIt seems that a couple of studies have investigated with simulations of the cost-effectiveness of PCV-13 usage in China. See for instance Mo et al. Pediatr. Infect. Dis. J. 2016 PMID 27753771, Tyo et al. Vaccine 2011 PMID 21745516.1,2 I would suggest to lookup for more publications of this type and to include a small paragraph summarizing the results in the introduction.\n\nMaybe it would also be useful to include a small paragraph on the serotype distribution in China before the introduction of PCV-13, see Lyu et al. Expert Rev. Vaccines PMID 28745918.3\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-221
|
https://f1000research.com/articles/7-1925/v1
|
11 Dec 18
|
{
"type": "Research Article",
"title": "Discipline-specific open access publishing practices and barriers to change: an evidence-based review",
"authors": [
"Anna Severin",
"Matthias Egger",
"Martin Paul Eve",
"Daniel Hürlimann",
"Martin Paul Eve",
"Daniel Hürlimann"
],
"abstract": "Background: Many of the discussions surrounding Open Access (OA) revolve around how it affects publishing practices across different academic disciplines. It was a long-held view that it would be only a matter of time for all disciplines to fully and relatively homogeneously implement OA. Recent large-scale bibliometric studies show however that the uptake of OA differs substantially across disciplines. This study investigates the underlying mechanisms that cause disciplines to vary in their OA publishing practices. We aimed to answer two questions: First, how do different disciplines adopt and shape OA publishing practices? Second, what discipline-specific barriers to and potentials for OA can be identified? Methods: In a first step, we identified and synthesized relevant bibliometric studies that assessed OA prevalence and publishing patterns across disciplines. In a second step, and adopting a social shaping of technology perspective, we studied evidence on the socio-technical forces that shape OA publishing practices. We examined a variety of data sources, including, but not limited to, publisher policies and guidelines, OA mandates and policies and author surveys. Results: Over the last three decades, scholarly publishing has experienced a shift from “closed” access to OA as the proportion of scholarly literature that is openly accessible has increased continuously. The shift towards OA is however uneven across disciplines in two respects: first, the growth of OA has been uneven across disciplines, which manifests itself in varying OA prevalence levels. Second, disciplines use different OA publishing channels to make research outputs OA. Conclusions: We conclude that historically grown publishing practices differ in terms of their compatibility with OA, which is the reason why OA can be assumed to be a natural continuation of publishing cultures in some disciplines, whereas in other disciplines, the implementation of OA faces major barriers and would require a change of research culture.",
"keywords": [
"Open Access",
"Open Science",
"Publishing",
"Scholarly Communication",
"Science Policy",
"Communication Technologies",
"Scientometrics",
"Meta-Synthesis"
],
"content": "Introduction\n\nAs a response to perceived limitations of the subscription-based model of scholarly publishing and propelled by technical possibilities provided by the internet, Open Access (OA) presents a new model of academic publishing1. OA takes different forms but generally offers free and unrestricted access to the outputs of academic research with relaxed constraints on reuse, as opposed to publications being “locked away” behind subscription paywalls2. Having gained global relevance, the potential implications of OA for academic publishing continue to generate debate in the academic community. Many of these discussions revolve around the question of how OA affects publishing practices in different academic disciplines3.\n\nThe foundation for OA was laid in high-energy physics when Paul Ginsparg established the arXiv open repository for preprints4. OA soon appeared to constitute an “inescapable imperative”5 for several reasons: first, OA gained early momentum based on a combination of grass-root advocacy initiatives promoting the unrestricted access to publications on the one hand and funding organisations, universities and national governments implementing OA mandates and policies that require scholars to make their outputs publicly accessible on the other hand6. Second, OA has the potential to enhance scholarly communication by speeding up the dissemination of research outputs, by expanding readership and by increasing the impact of research outputs7. From an information-processing perspective, scholars across all fields should see these benefits and use OA communication channels uniformly5. These trends suggested that it would only be a matter of time for all academic disciplines and fields to fully adopt OA and to converge on a stable set of relatively homogeneous OA publishing practices8. In contrast to these expectations, recent bibliometric studies show that academic disciplines vary considerably in terms of their OA publishing practices9,10.\n\nBibliometric studies investigating disciplinary OA publishing practices are in large part descriptive and, as such, do not analyse the mechanisms that shape discipline-specific OA publishing practices. This limitation becomes relevant as vast amounts of resources and efforts are committed to the development, maintenance and advancement of OA communication channels. In the absence of a valid theory of how academic disciplines adopt OA, resources may be dedicated to ventures that are not sustainable. We aim to address this by answering the following questions: (1) How do different academic disciplines adopt and shape OA publishing practices? (2) What discipline-specific barriers to and potentials for OA publishing can be identified? In order to answer these questions, we first synthesise relevant bibliometric studies that were aimed at assessing the prevalence and patterns of OA publishing practices across academic disciplines. Adopting a social shaping of technology perspective, we then develop an analytical framework that consists of socio-cultural and technological factors that generally shape publishing practices. We apply this analytical framework to the case of OA publishing and examine evidence on the forces that represent barriers to and potentials for OA, causing OA publishing practices to differ across disciplines. Doing so, we examine and aggregate evidence from a variety of primary data sources including, but not limited to, OA mandates and policies, infrastructures of scholarly communication technologies and author surveys.\n\n\nMethods\n\nThe objective of our review is to identify and synthesize large-scale bibliometric studies on the prevalence and patterns of OA publishing across academic disciplines. Such studies usually analyse similar samples of academic publications, including data from Web of Science (WoS), Google Scholar (GS) and Scopus, but employ different methods for identifying disciplinary publishing practices within these databases. This relates to definitions of OA, included OA routes, covered publication years and employed search strategies for OA full texts. For this reason, we conducted a meta-synthesis. The aim of a meta-synthesis is to qualitatively integrate, compare and analyse methodologically heterogeneous studies, thereby allowing the emergence of interpretive themes11. In this study, we synthesised the results from bibliometric studies to identify patterns of OA publishing practices across academic disciplines. The search was pre-planned and comprehensively, as it aimed to seek all available studies. No date limits were employed. The searches were conducted in August to October 2018. Bibliometric studies were searched in a systematic way. This involved, first, the querying of the online data bases ScienceOpen, Scopus, WoS and GS. The search was conducted using the following search string: “Discipline” AND “Publish*” AND “Open access” OR “OA”. The selection of the search terms was based on the topic literature on scholarly communication. Second, reference lists and bibliographies of all included studies were evaluated manually for additional publications. Having identified key experts within the field, their GS profiles were also searched for material. In an initial screening stage, two independent reviewers screened titles and abstracts of studies and decided on whether to include respective studies in the review. Studies were excluded that did not meet our selection criteria, as outlined in Table 1. This procedure resulted in a total of 11 studies. In a second screening stage, we assessed the full text of the included studies. In order to gain the data of interest to our review, we analysed the “Results” sections of primary studies and extracted data on reported proportions of publications that were OA, including both the overall OA proportions and the relative uptake on OA routes.\n\nOur goal in this section is to explain the patterns of OA publishing practices that we observed in the previous section. To do this, we performed a narrative review of the mechanisms and factors that shape OA publishing practices in different academic disciplines. Thereby, we aim to identify discipline-specific barriers and potentials for OA. We recruited an interdisciplinary team of researchers covering the broad academic disciplines natural and technical sciences, medicine and health-related sciences, social sciences and law, arts and the humanities. Each co-author of our team examined evidence on factors that shape OA publishing practices within their own areas of research training. In doing so, we did not perform a systematic review of the literature. Instead, we developed an analytical framework of socio-cultural and technical factors that generally shape publishing practices. Each co-author used this framework as a tool for identifying the socio-technical mechanisms and factors that shape OA publishing practices within their own discipline. The evidence included in this review was identified and selected through queries of online databases, including ScienceOpen, Scopus, WoS and GS. Reference lists and bibliographies of relevant studies were evaluated manually for additional evidence. Having identified key experts within the field, their GS profiles were also searched for material. Each co-author contributed original content on OA in their discipline and participated in the reviewing and editing process.\n\n\nResults\n\nThe characteristics of the studies included in our review are presented in Table 2. In general, studies were concerned with the questions of (1) how much of the scholarly literature in a academic discipline is openly accessible, and (2) via which OA route scholarly outputs are made openly accessible. Earlier studies analysed random samples of academic publications from bibliometric databases, such as Scopus or WoS, whereas more recent studies examined these databases in full. Making use of automated web search strategies, studies assessed whether openly accessible versions of sampled scholarly publications could be found on the web, for example through GS. On this basis, studies determined OA levels and the relative uptake on different OA routes across disciplines. Earlier studies distinguished between Green OA, which refers to articles published in subscription-based journals, but for which either the accepted or the published version can be retrieved from an open repository, and Gold OA, which describes articles published in OA journals, that is, journals in which all articles are openly accessible. More recent studies also include Hybrid OA, which refers to articles free under an open license in a subscription journal, and Bronze OA, which describes articles free to read on the publisher page without an open license12 1.\n\nTable 3 shows the main findings of the studies included in our meta-synthesis. Looking at the overall prevalence of OA for all disciplines, we see that the system of academic publishing has experienced a shift from “closed” access to OA: OA levels have increased steadily across all disciplines, from 20.4% of all scholarly outputs reported as OA in 200815, to 23% in 20109 and more than half of all scholarly outputs being OA in publication years later than 2010: 53.7% for publication years 2011 until 201316, 54.6% on average in years 2009 and 201417, 66& for publication years between 2009 and 201718 and 55% in 201419. Two studies determined the prevalence of OA to be less than 50% for publication years later than 2010: Piwowar et al. determined that on average 36.1% of the scholarly literature was made OA in the time period between 2009 and 2015 and Bosman and Kramer found this share to be 29.4% for 201612,20. Looking at how different disciplines implemented OA over time, we can distinguish between three phases. Dated between the early 1990s and the mid to late 2000s, the first phase can be characterised as a phase of formation: A few fields related to the natural and technical sciences took on a pioneering role in implementing OA, amongst these particularly mathematics (reported OA levels of 25.6% and 42% in 2008 and 2010, respectively1,9) and physics and and space-related research fields (OA levels of 32.9% for earth sciences and 23.5% for physics & astronomy in 2008, and 37% for earth & space and 27% for physics in 20101,9). An exception to this are the fields engineering and chemistry, which feature OA prevalence rates that consistently are lower than all natural and technical sciences and lower than most other disciplines, including the social sciences and the humanities1,9. The social sciences were also fast in embracing OA, featuring OA prevalence levels only slightly below those reported for the natural and technical sciences (16% OA in sociology in the time from 1992 to 2003, followed by economics with 13.5% OA and business with 9% OA21; 23.5% OA and 37% OA observed in the social sciences for publication years 2008 and 2010, respectively1,9). Medicine and health-related research fields were substantially slower in implementing OA than most natural and social sciences (OA levels of 6.2% in medicine between 1992 and 200321; 21.7% for medicine and 15.2% for other areas related to medicine in 20081; 17%, 14% and 12% OA reported for health, clinical medicine and biomedical research in 2010, respectively9). As such, medicine and health-related fields implemented OA to an even smaller degree than the humanities have in the early years of OA (19% OA reported for humanities in 20109). The second phase of OA is dated between the mid 2000s and the mid 2010s and can be characterized as a period of transformation. In medicine and health-related research fields, OA uptake increased substantially, causing OA levels in these fields to equal or surpass OA prevalence in the social sciences and humanities (26% OA determined for medicine for publication years until 2013, while 24% OA showed for arts and humanities22; 59.7% OA in health sciences between 2004 and 2014, while 60.8% of publications in social sciences were OA16). For the early period of this phase, OA levels in the natural and technical sciences remained well above those observed in other disciplines (Observed OA levels were 50% in computer sciences, 35% in both geo-sciences and physics, 29% in environmental sciences and 27% in mathematics in publication years until 201322; 60% OA in physical sciences in the time from 2004 to 201416). Because of higher OA growth rates in medicine and health towards the end of this phase, these fields soon overtook the natural and technical sciences in embracing the idea of OA. Particularly biomedical research took on a leading role in embracing OA (70.6% OA in biomedical research, 67.6% OA in mathematics & statistics, 66.2% for biology, 59.4% for physics & astronomy and 58.8% for earth and environmental sciences, closely followed by public health & health services and clinical medicine with OA levels of 57.2% and 56,3%, respectively, in the period from 2011 to 201310). During this period, the gap between the natural and technical sciences and medicine on the one side and the social sciences and humanities on the other side widened. The humanities in particular published research outputs to lesser degrees OA than other disciplines (35.0% OA in arts, humanities & social sciences, 34.7% in philosophy & theology, 34.4% in historical studies for publication years 2011 to 2013)10. The third phase of OA can be dated after the early 2010s and is a phase of stabilisation, in which differences in the OA publishing patterns across disciplines have become established. Studies consistently show that medical and health-related research fields are taking the leading roles in embracing OA, featuring OA uptake levels that are well above those reported for other disciplines (Reported OA levels are 60% in medical and life sciences on average for 2009 and 201417; 59% for health sciences in 201419; 58.5%, 47.8% and 41.8% for biomedical research, clinical medicine and health in publication years from 2009 to 2015, respectively12; 41.7% for life sciences and biomedicine in 201620 and 85%, 79% and 73% for biomedical research, clinical health and health in publication years from 2009 to 2017, respectively18). The medical sciences are closely followed by disciplines from the natural and technical sciences (50% OA for natural sciences on average in 2009 and 201417; 55% OA for natural sciences in 201419; 52.7% OA for mathematics, 40.4% OA in earth and space, 32.7% OA in biology and 31.6% OA in physics between 2009 and 201512; 14.8% for physical sciences / technology in 2016;20; 57% OA for mathematics, 56% OA for earth and space, % 56% OA for physics and 51% OA for biology in publication years from 2009 to 201718). OA uptake in the social sciences is close behind the natural sciences (Reported OA levels are 49.9% for social and behavioural sciences in 2009 to 201417; 55% for economic and social sciences in 201419; 25.1% in social sciences between 2009 and 201512; 17.3% in social sciences for 201620; 39% for social sciences between 2009 and 201718). Law, arts and humanities show the lowest OA uptake across all disciplines (OA prevalence rates determined to be 32.3% for law, arts and humanities between 2009 and 201417; 24% for arts and humanities in 201419; 13.9% for arts and humanities in 201620).\n\nLooking at the relative uptake of OA routes for all disciplines, we observe that most OA is published via the Green route, that is, published as journal articles for which the accepted or the published version can be retrieved from an open repository. Gold OA journals are also of importance for scholarly publishing, even though the relative uptake on Gold OA remains well below Green OA for most publication years (Relative uptake levels were 11.9% Green OA and 8.5% Gold OA in 2008, respectively,1, 21% Green OA and 2% Gold OA in publication years from 2005 to 20109, 5.9% Green OA and 12.1% Gold OA between 2011 and 201310, 10.8% Green OA and 7.3% Gold OA on average in publication years 2009 and 201417, 8.8% Green OA and 49.4% Gold OA in publication years between 2004 and 201416, 31% Green OA and 23% Gold OA in 201419 and 11.5% Green OA and 7.4% Gold OA in publication years between 2009 and 201512). Studies that also assessed the relative uptake on Hybrid OA and Bronze OA have revealed, that, first, Hybrid OA generally is of little importance for scholarly publishing, with 1% or less of all scholarly outputs being published as articles free under an open license in subscription journals. Second, the importance of Bronze OA is comparable to that of Gold OA. Relative uptake on Bronze OA was determined to be an average 13.2% for publication years 2009 and 2014 and 12.9% for publication years from 2009 to 201512,17. Looking at the relative uptake on OA routes by discipline, we observe that there is little consistency in reported uptake levels across the studies included in our review, which likely is due to methodological differences in how studies determined how much of the scholarly literature in a specific discipline is published through different OA routes. Nevertheless, we are able to determine the relative importance of the different OA routes for each discipline: For the medical sciences, we observe that publication in pure OA journals (Gold OA) plays a more important role for making research findings openly accessible than both the archiving of articles in repositories following publication in subscription journals (Green OA) and the publication of articles free under an open license in subscription journals (Hybrid OA). Two more recent studies have revealed that the publication of articles free to read on the publisher page without open license (Bronze OA) also is of substantial relevance for OA in the medical sciences, featuring similar prevalence levels as Gold OA. For the natural and technical sciences, we see that there are substantial differences in the OA publishing patterns between different fields: scholars in physics, mathematics, astronomy and biology make large shares of their research outputs openly accessible through the Green route of OA, followed by Bronze OA, Gold OA, and, with some distance, Hybrid OA. In contrast, for scholars in chemistry and biology, Gold OA journals are of greater importance than any other OA route. For scholars in the social sciences, Green OA is of greater importance for OA publishing than Gold OA, Bronze OA and Hybrid OA. In the humanities and law, scholars make research outputs openly accessible predominantly through publication of articles in Hybrid OA journals, followed by Green OA, Bronze OA and Gold OA.\n\nPrevious studies have analysed discipline-specific publishing practices from a range of perspectives. In general, these perspectives originate from science and technology studies (STS), which look at how society, politics and culture shape research and technological innovation, and vice versa. Depending on their understanding of the direction of this relationship, these perspectives can be located on a scale that ranges from technological determinism at the one extreme to social constructionism of technology (SCOT) at the other extreme23. These perspectives emphasize particular sets of criteria as relevant for analysing publishing practices while other sets of criteria are considered irrelevant or ignored5. Technological determinism suggests that technology is the driving force behind social and cultural change24. Studies adopting this perspective accordingly focus on the infrastructures and technical aspects of scholarly communication channels in explaining how OA is implemented across disciplines. Social and cultural factors are believed to be of less or no relevance in explaining the emergence of OA5. SCOT perspectives view technology as a social phenomenon constructed by the society producing and using it. In order to analyse OA publishing patterns, one would have to first understand the social relations within which respective technologies are used25. Both positions have been shown to suffer from limitations in explaining scholarly publishing practices2. The so-called “social shaping of technology” (SST) perspective that takes an intermediate standing between these extremes proves to be more useful for analysing OA publishing practices. SST is a theoretical stance that conceives the relationship between technology and society as one of mutual shaping26. Instead of evolving according to an inner technical logic or a single social determinant, technology is believed to be a social product patterned by the conditions of its creation and use27. Central to technical change are choices made by social actors and groups during the generation and implementation of new technologies. This process involves a set of conscious and unconscious choices between different technical options28. Which options social actors select is affected by both technical considerations and a range of social and cultural aspects. Thus, social choices influence the contents of technologies. At the same time, technologies have social implications as they shape human action and behaviour26. Following this, scholarly publishing practices can be understood as socio-technical ensembles: the ways in which scholarly outputs are published is affected by the operational choices made by scholars during the creation, implementation and use of respective communication technologies. These operational choices are influenced by both technical considerations and socio-cultural aspects. As communication technologies are implemented and used, they in turn affect the ways in which scholars communicate and disseminate their research findings. It follows that, in order to explain discipline-specific OA publishing practices, it is necessary to examine the socio-cultural and technical factors that affect publishing choices within particular disciplines. Based on these assumptions, we have developed an analytical framework that places focus upon technical factors and socio-cultural factors alike when analysing patterns of OA publishing practices. The analytical dimensions entailed in this framework are illustrated in Table 4.\n\nInitially, medicine and health-related disciplines were reluctant to adopt OA publishing, resulting in OA levels to be well below those observed in the natural and social sciences. From the mid-2000s onwards, however, the uptake on OA increased substantially and particularly biomedicine and clinical medicine took on leading roles in embracing OA. Research outputs are predominantly made OA by publication in Gold OA journals, whereas Hybrid OA, Bronze OA and Green OA are of little importance for these disciplines. Factors facilitating OA can be identified as strong OA mandates combined with either funder-operated repositories or available funding for article processing charges (APCs), the richness in high-quality and prestigious OA journals and the wide circulation of publications in these outlets. A major barrier to OA in the medical sciences are authors’ concerns over the quality of peer review in OA journals, which is related to the emergence of fraudulent journals and publishers.\n\nA) Author behaviour and attitudes – Several surveys and interview studies have shown that in biomedicine and the life sciences, a large majority of authors support OA publishing, but the reputation of journals, their impact factor, and the quality and speed of peer review are more important factors determining the choice of publication outlets than the OA status29–31. For example, in 2004, Sara Schroter and colleagues interviewed authors who submitted articles to the BMJ. Almost all authors supported the concept of OA, but many were concerned about poor quality research being published for a fee, and OA was not a factor of importance when selecting a journal30. More recently, the 2014 international author survey conducted by publisher Taylor & Francis showed that investigators working in Science, Technology and Medicine (SEM) mentioned wider circulation than publication in a subscription journal as an advantage of OA, but were strongly against to the use of their work for commercial gain without their explicit permission32. Authors expected rigorous peer review and rapid publication in return for paying for OA publication32,33. Surveys among academics from lower income countries indicate that the funding of APCs is an important concern34,35. A study from India found that the most important factors influencing the selection of medical or dental journals were that the journal is indexed in widely used bibliographic databases, has an online submission system, a satisfactory impact factor and peer review, and that APCs are affordable36. The importance of affordable APCs may explain why authors from resource-limited settings are over-represented among publications in fraudulent journals that charge small fees but do not provide proper peer review or add value through editing37.\n\nB) Publisher behaviour and policies – As private profit-oriented companies, most traditional publishers are driven by maximizing income to satisfy their shareholders3. Consequently, as the OA model is unlikely to generate the level of income and profit that can be achieved with the subscription model, few commercial medical publishers have converted their subscription journals to OA. This also applies to academic or professional societies29. Policies on prior publication remain tight for most of these journals. Some journals have now moved to allowing their authors to self-archive submitted manuscripts without an embargo period, while self-archiving of accepted versions of a publication remains subject to a standard embargo period of 12 months. Pioneers among OA medical journals include the Journal of Clinical Investigation, which in 1996 became the first major journal to be freely available on the web. Of note, publication in the journal was free to authors initially, but APCs were introduced after the journal lost 40% of its institutional subscribers38. The BMJ followed suit in 1998, but moved some content (including editorials and education and debate articles) behind a pay wall in 200539. The number of OA journals increased considerably from 2000 onwards, with the arrival and rapid growth of OA publishers such as the not-for-profit publisher Public Library of Science (PLoS) or the commercial publisher BioMedCentral (BMC). The launch of OA journals by major biomedical research funders40–42 and the emergence of mega-journals are other factors that have influenced uptake of OA publishing in medical research fields43.\n\nC) Infrastructure of scholarly communication – OA publishing in the medical sciences focuses on Gold OA journals and only a small number of OA institutional and subject repositories has emerged. This can be explained as follows. First, sufficient funding is available for publication in Gold OA journals. Second, journal publications are of central importance in academic hiring and promotion decisions within the medical sciences. Third, there is a large number of high-quality Gold OA journals for authors to publish their research in. The uptake on open repositories in general is low, but an exception to this is the PubMed Central (PMC), which archives full-text scholarly articles and plays a central role in the medical and life sciences. PMC has experienced rapid growth in the late 2000s as the National Institutes of Health (NIH) introduced an OA policy that mandates its grantees to deposit the final peer-reviewed version of an article based on NIH-funded research in PMC. The embargo was initially 12 months after publication, but was later shortened to 6 months44 and journals have since moved to be compliant with this Green OA mandate. Submissions into the PMC undergo indexing and formatting procedures, which produces advanced metadata and unique identifiers45. Of interest, even though not of the same relevance as PMC, is also the PeerJ Preprint section, which allows authors to submit preprints and postprints from the biological and medical sciences.\n\nD) Structural and institutional factors – The main type of work products in the medical sciences are journal articles. Like the natural sciences, research in the medical sciences and related fields in most parts is funded by project-specific grants, which makes it fairly easy to integrate processing charges for publication in OA journals into existing funding structures. Further, medical research is in large part funded by third-party funding, for example by the World Health Organization (WHO) and the Wellcome Trust. These organizations have strong OA mandates while, at the same time, providing both convenient open repositories for depositing articles and sufficient funds for covering processing charges for publication in OA journals46.\n\nE) OA mandates and policies – Evolving national and institutional OA policies, OA mandates by major funders of (bio-)medical research and the availability of funding for APCs have accelerated the uptake of OA publishing in the medical and life sciences. A substantial number of national governments have moved to require scholars in the medical and life sciences to make their articles OA if based on publicly-funded research by either publishing in OA journals or by making publications OA by depositing the accepted or the published version of an article in a repository. Usually, scholars are granted embargo periods of 6 or 12 months to comply with the latter18. Besides national governments and research institutions, major funders of medical research play an active role in promoting OA. Besides the NIH, this includes the WHO and the Wellcome Trust. Since 2014, journal articles and book chapters based on WHO-funded research have to be published in either an Gold or Hybrid OA journal or in a subscription journal that allows the author to deposit the accepted version in PMC no later than 12 months after publication47. Similarly to the NIH, the Wellcome Trust requires articles to be published in OA journals where a journal makes this option available and to be deposited as the accepted version in an open repositories no later than 6 months after publication. Both funders state that they will withhold or suspend payments if articles are not made OA. Both funders provide repository infrastructures (PMC for NIH and PMC or PMC Europe for Wellcome Trust) and funds for covering APCs46. In contrast to the USA, the policy environment in the UK favoured gold and hybrid OA, with particularly high uptake in the life sciences and rapidly increasing costs48.\n\nFor most publication years, the natural and technical sciences show the highest OA prevalence rates amongst all disciplines. There are, however, substantial differences in the OA publishing patterns between different subdisciplines of the natural and technical sciences. Journals in the fields of physics, mathematics, astronomy, information technology and biology were the early pioneers of OA and continue to make large shares of their research outputs OA. In contrast, engineering and chemistry feature OA prevalence rates that are consistently much lower than in other fields of the natural and technical sciences and even slightly lower than OA levels observed in the social sciences and humanities. While Green OA seems to be of central relevance for OA publishing within physics, astronomy, biology, information technology and mathematics (followed by Bronze, Gold and, by some distance, Hybrid OA), scholars in chemistry and biology make larger shares of their research OA through publication in Gold OA journals than in open repositories. Factors facilitating OA in the natural and technical sciences can be identified as the long-existing culture of preprint distribution, availability in funding for APCs and high levels of awareness of and familiarity with OA publishing. Barriers to OA are concerns about the quality of OA journals and high degrees of industrial integration in some fields.\n\nA) Author behaviour and attitudes – The distribution of preprints has a long tradition in many fields related to the natural sciences, particularly in physics, mathematics, astronomy, information technology and biology, where scholars commonly share their manuscripts before submitting these for publication to journals. Before it was possible to make documents available electronically, a paper-based culture of preprint distribution developed in the 1960s, especially in high-energy physics49. With the emergence of the Internet, scholars began sharing electronic versions of their preprints informally via electronic mail and when Paul Ginsparg established the open repository arXiv in 1991, scholars started making their preprints openly accessible through centrally self-archiving them in arXiv4. Surveys have revealed that, to scholars within these fields, rapid publication, high visibility and large readership appear to be the most important factors when it comes to choosing a publication outlet, and that scholars associate these features with depositing preprints in open repositories33,50. Adding to this, scholars in the natural sciences generally show high levels of familiarity with the concepts of OA in general and Green OA in particular15,51. As a consequence, Green OA has become the most popular way of making research outputs OA in physics, mathematics, astronomy, information technology and biology. Publishing in journals (closed-access and Gold, Hybrid or Bronze OA) is less prominent for scholars within these fields. In contrast, scholars in chemistry and engineering value publication in journals over self-archiving in repositories, which is the reason why Gold OA plays a bigger role than Green OA in these fields50. Despite the preprint culture in some of the natural sciences, 40% to 50% of all research outputs overall remain closed-access today. Chemistry and engineering show particularly low uptake levels on OA. This might be due to the fact that scholars within these fields still have concerns about the quality of peer review in OA journals and are concerned that this might translate into low-quality publications in these outlets. Consequently, OA journals within the natural sciences have not yet been able to match the reputation of subscription journals52.\n\nB) Publisher behaviour and policies – Commercial publishers as well as learned societies in the natural and technical sciences have been slow in embracing the idea of OA. This relates to two factors: First, publishers face a potential loss of revenues in switching from a subscription model to an APC model, as has been shown in a number of market analyses53,54. Second, general concerns about the quality of OA journals are not only shared by scholars but also by publishers and learned societies52. As a result, most of the major commercial publishers, as well as learned societies in the natural sciences, have been reluctant to either convert their existing subscription journals to OA and to set up new OA journals. An exception to this are few large publishing houses that have started setting up new OA journals in disciplines that do not have a culture of preprint distribution, such as chemistry or engineering. In disciplines where there is a culture of preprint distribution, publishers have started relaxing policies on prior publication and enable manuscripts deposited in repositories to be directly submitted to their journals52.\n\nC) Infrastructure of scholarly communication – In physics, mathematics, astronomy, information technology and biology, scholars are used to sharing their research outputs openly making use of open repositories, particularly arXiv. Originally established within high energy physics, arXiv now is used by scholars in most fields of the natural sciences and its concept has eventuated in a number of discipline-specific repositories in other fields, including the social sciences. Even though repositories do not employ formal mechanisms of quality control, scholars within the natural sciences use them to first, disseminate their research outputs without publication delays, and second, stay informed about ongoing research within their fields4. In the light of this publication culture, relatively few OA journals have emerged within these fields. In fields where there is a smaller culture of self-archiving in repositories, most particularly in chemistry and engineering, the number of OA journals has grown slowly but steadily in recent years. These journals cover a variety of specific subject areas, are peer-reviewed, and, for the most part, are published in English. Exemplary journals in engineering are the International Journal of Antennas and Propagation, the Journal of Engineered Fibers and Fabrics, Journal of Scientific and Industrial Research and Thermal Science55. Chemistry journals that enjoy popularity are the Archive for Organic Chemistry, Beilstein Journal of Organic Chemistry, Chemistry Central, Catalysts and ChemistryOpen52.\n\nD) Structural and institutional factors – The main types of work products in the natural and technical sciences are journal articles, electronic preprints and conference proceedings, which are published records of conferences, congresses or other meetings. Researchers from the natural sciences have reported that the process of self-archiving electronic preprints and conference proceedings is little time-consuming and that they generally experience little difficulties in making research outputs OA using open repositories50. In addition, and similar to the medical sciences, research in the natural sciences is in large parts funded by project-specific grants, which would make it fairly easy for scholars to integrate fees for publication in Gold or Hybrid OA journals into existing funding structures. A structural factor that limits the uptake on OA within the natural and technical sciences is that some of these fields, particularly chemistry and engineering, are industry-oriented. This adds to the fact that, particularly within engineering, the focus is rather national than international as products developed by engineers are, for the large part, produced for domestic markets. As a consequence of these factors, large numbers of publications within these fields are more practice- than science-oriented and are published in closed-access journals that are partly financed by advertising55.\n\nE) OA mandates and policies – Reflecting the ambition to make research outputs OA, there are strong OA mandates for the natural and technical sciences. Usually, these fields are subject to similar OA requirements as the medical sciences: scholars are usually required to make their outputs OA if based on publicly-funded research by either publishing in OA journals or by depositing the accepted or the published version of an article published in subscription journal in a repository. By default, scholars are granted embargo periods of 6 or 12 months to comply with the latter18. Besides national and international funding agencies, CERN and the Sponsoring Consortium for Open Access Publishing in Particle Physics (SCOAP) play leading roles in promoting OA. SCOAP is an international partnership of funding agencies, research centers and libraries that was launched with the aim of providing funding for the conversion of high-energy physics journals from a subscription model of publishing to OA. Within this scheme, libraries and research centers either pay reduced subscription fees for participating journals or stop paying altogether. Saved monies feed into a central fund, which is used to pay publishers up front to publish OA articles56. Doing so, the initiative enables scholars to make their research outputs OA without straining their own research funds. By 2014, five journals had been converted within the framework of SCOAP49. The OA policy of CERN requires its scholars to publish their articles, wherever possible, in journals covered by SCOAP. When circumstances require publication in journals that are not covered by SCOAP, the APCs must be covered by funds from outside the CERN Budget, for example through EU projects or by other institutions. Where this is not possible, authors may request special permission and funds from CERN57.\n\nOverall, the OA uptake in the social sciences is higher than in most disciplines of the humanities, but remains below the medical and natural sciences. Publishing in Gold OA journals plays a less important role than the archiving of publications in institutional and subject repositories following publication in a subscription journal. For scholars within the social sciences, open repositories appear to be of central importance for making research outputs openly accessible, closely followed by publication in Gold OA journals, and, with some distance, Hybrid and Bronze OA. The low uptake on OA is due to a variety of reasons, including low levels of awareness, concerns about quality and prestige of OA journals, the central role of monographs for career advancement and difficulties in accessing funding for APCs and Book Processing Charges (BPCs). Having said that, the social sciences are currently experiencing a cultural shift towards conducting science more openly, which manifests itself in an increasing embracement of OA.\n\nA) Author behaviour and attitudes – Author surveys consistently have revealed that the awareness of OA publishing is lower for the social sciences than for the medical and natural sciences, and that OA publication outlets have not yet fully become part of the workflow for social scientists51. The knowledge of OA journals and repositories however appears to grow amongst social scientists with particularly young researchers reporting high levels of OA awareness and engagement58. Most social scientists support the idea of OA in principle, but stringent quality control, further improvement of the manuscript before publication and journal prestige still appear to outweigh OA in authors’ journal selection criteria59,60. As a consequence, OA publishing activity remains low for the social sciences. This is also due to the fact that some social scientists and their learned societies are still opposed to OA, which relates mainly to concerns about quality of peer review and editorial services in OA journals61. Relevant to the appreciation of OA in the social sciences is also the importance attached to monographs. While in the natural and medical sciences, the large part of research findings is disseminated via journal articles, the monograph has a central place in the culture and ecology of publishing in most of the social sciences and is highly relevant to career advancement60,62. Monographs have been shown to be less likely to be published OA. Amongst other factors, this relates to authors’ concerns over restricted editorial services and doubts whether unestablished OA publishers and formats are able to translate their effort in writing a monograph into reputational gain within the scientific community63.\n\nB) Publisher behaviour and policies – Few publishers in the social sciences have decided to convert their existing subscription-based journals to OA or to set up new OA journals. Key academic journals in the social sciences remain closed access. Amongst other factors, this relates to publishers fearing that their academic authors will not be able to access funding for APCs or that switching to an APC model will result in a loss of prestige – both of which are main factors affecting authors’ choice of publication venue60. For some journals, such as the Historical Social Research or the Zeitschrift für Soziologie, it has become common practice to make their contents automatically OA after an embargo period of two years either by enabling access to their articles on their own website or by depositing them in an OA repository64. In addition to this, a large variety of new economic models of OA publishing has emerged that offers viable alternatives to author-payment model in the social sciences and humanities. To name only two, this includes Knowledge Unlatched (KU) and the Open library of Humanities (OLH). OLH is based on a business model that is called “Library Partnership Subsidy” and which asks libraries to pay a relatively small annual subscription fee to enable OA to scientific publications. The model originally was aimed at journals in the humanities and social sciences, but has been expanded to monographs63. The goal of KU is to create a financially sustainable route to OA for monographs through a global co-operated model where libraries use their existing acquisition budgets to enable OA to monographs63. Another innovative business model of OA publishing that has gained some popularity in the social sciences and humanities is the so-called “freemium” model. This model makes HTML versions of articles and books openly available to everyone, while PDF and ePub formats are accessible only to subscribing libraries and research institutes65. One well-known example of this is OpenEdition. While long-term access to research outputs is questionable within these models, OpenEdition and others managed to convince otherwise conservative publishers to create open versions of their journal volumes and monographs66.\n\nC) Infrastructures of scholarly communication – The social sciences are currently experiencing a considerable growth of open repositories, resulting in authors being able to choose from more than 200 different OA repositories, the most of which are institutional or subject repositories67. While subject repositories have become a fairly established part of the workflows for social scientists, institutional repositories are less often used and predominantly host faculty working papers and theses. Prominent examples of subject repositories are the Social Science Research Network, the Social Science Open Access Repository and SocArXiv. Because OA preprint repositories do not employ peer review, however, social scientists have been slow to adopt Green OA. Gold OA journals are of even less importance for the social sciences. Key academic journals in most countries remain closed access68. The few existing OA journals in large part are restricted to highly specified sub-disciplines with limited impact and small readership. One notable exception to this was the launch of SAGE Open in 2011, which has brought to the social sciences the OA mega journal model already popular in the natural and medical sciences69. In addition to this, a number of OA journals were launched by academic or professional societies, such as Socius: Sociological Research for a Dynamic World launched by the American Sociological Association in 201650.\n\nD) Structural and institutional factors – Similar to most disciplines of the humanities, monographs are one of the main work products in the social sciences and highly relevant for academic promotion and career advancement. Besides author concerns over prestige and standards of editorial services of OA monograph publishers, the high costs and procedural complexities associated with producing monographs are important factors restricting the uptake on OA of monographs in the social sciences70. In addition to this, social scientists have reported to face significant difficulties in access to grant funding for both APCs and BPCs, as most research in the social sciences is not done by means of project-specific funding that is commonly used to compensate APCs in the natural and medical sciences31.\n\nE) OA mandates and policies – Scholars in the social sciences face similar OA requirements as scholars within the natural and medical sciences do. Some special regulations can be identified, however. First, monographs are generally not included in OA mandates. Most public funders limit themselves to recommending OA for monographs. One of the few exceptions to this is the SNSF, which demands the OA publication of monographs and provides respective funding for BPCs63,71. Second, the social sciences commonly are granted longer embargo periods for the archiving of a journal article after publication in a subscription journal. While embargo periods of 6 or 12 months are the default for the natural and medical sciences, social scientists usually have to deposit journal articles in institutional or subject repositories after up to 12 or 24 months following publication18,72.\n\nGenerally speaking, OA uptake in the humanities is lower than in most areas of the natural, medical and social sciences. This is partly due to the fact that these disciplines exist in a “dry climate” of funding for gold OA models that rely on APCs73. Low uptake is also due, though, to the fact that the monograph plays such a central role in many humanities disciplines, but the funding challenges for open access to such outputs remains an unresolved problem at scale74,75. Hybrid OA is of central importance for the humanities, followed by Green OA, Bronze OA and Gold OA. Given that the humanities focus on the study of human cultures and artforms, it is, though, nonetheless surprising that more humanists do not seek to reach general public audiences through broader availability of their research work.\n\nA) Author behaviour and attitudes – As in many academic fields, authors operate within a symbolic economy of prestige that is usually among the prime motivations in choice of publication venue76. The relative prestige of publications is determined by a scarcity correlation (usually achieved through peer review) with the shortage of evaluative labour on hiring, tenure, and grant panels, although most humanities fields use an informal hierarchy of publications rather than quantitative measures such as the Impact Factor75. Although institutional signups to the San Francisco Declaration on Research Assessment may help to change this through a shift to evaluation at the article level, the focus on the Impact Factor in that declaration may make it harder to alter evaluative cultures in these disciplines. Further, academics and learned societies in the humanities disciplines have often been opposed to open access, for a variety of reasons that range from concerns over misunderstanding, worries about open licensing and plagiarism, or fears for the standing of their members77,78. In addition to this, humanities scholars show fairly low levels of awareness of OA and potential OA publication outlets in their fields79. That said, there are signs of a cultural shift with new economic models that do not rely on author payments, such as KU, the OLH, Open Humanities Press, Open Book Publishers, Punctum Books, and others appearing to have some traction with at least some humanities scholars. Although it is tempting to posit that humanities scholars are simply less driven by technological change than their counterparts in scientific disciplines, and thereby less inclined towards digital (and, therefore, open) publishing solutions, this is a generalized assertion that is hard to substantiate.\n\nB) Publisher behaviour and policies – The main concern driving humanities publishers is ongoing sustainability of their operations. In switching to an APC or BPC model, often undifferentiated from scientific publications, publishers fear that their academic authors will not be able to pay. It is also clear that highly selective publication models, which are common in the humanities, are more difficult to run, economically, on an OA basis. Hence there is little movement towards a fully gold OA ecosystem, although it is unclear what impact the recently announced pan-European initiative, Plan S, may have upon this. That said, most humanities publishers are compliant with green OA mandates, such as the UK’s REF policy80. On the other hand, it is also the case that some humanities scholars have argued that a longer citation half-life (particularly for monographs) should translate to longer embargo periods within these disciplines, although this does not necessarily match up to sales half-lives81. Despite some disciplines having healthy cultures of offline working paper circulation (philosophy, for instance), preprints have not taken off in the humanities and policies on prior publication remain tight, especially in the most prestigious venues.\n\nC) Infrastructure of scholarly communication – In addition to institutional repositories, there has been a growth in recent years of OA subject repositories, such as the MLA Commons, which is operated by one of the largest subject associations in the humanities. There has also been a prominent culture, for many years, of scholar-led OA journal and book publications76. Postpublication peer-review remains rare and usually elicits scant participation without active intervention, with a few notable exceptions and experiments82,83. There is no infrastructure at a comparative scale to arXiv in the humanities disciplines. Furthermore, for long-form reading, print remains a crucial resource and scholars often report that they do not wish to read works of 80,000-words length in a purely digital format.\n\nD) Structural and institutional factors – The high costs of producing monographs are a key structural factor that currently limits OA in the humanities62,70. Further, most research work in the humanities does not receive project-specific funding, making it difficult to integrate processing charges into a grant. That the humanities disciplines are often of lesser importance in institutional hierarchies also means that it can be difficult to secure funding for articles. The slow cycle of producing long-form outputs is also problematic for OA, as the time investment (and hoped-for credit on publication) is greater than those of a journal article, leading scholars into more conservative prestige-seeking behaviours. There are also substantial challenges around third-party rights and re-use of images, particularly within disciplines such as Art History. Museum policies on licensing have not kept pace with digital publication practices and still often rely on “number of copies” as a metric determining pricing for re-use. Under such a paradigm, it can be difficult (or very expensive) to negotiate re-use rights for unlimited online dissemination. Finally, some disciplinary spaces, such as creative writing, have developed outward facing cultures that rely on sales. Creative writing scholars are often assessed on whether they can produce a “bestselling novel”, which works poorly under an OA model. The production of such artifacts may, however, have a research process behind them and various institutional policies will regard such objects as scholarly undertakings. The extent to which such work should be exempted from OA mandates remains, therefore, an ongoing debate.\n\nE) OA mandates and policies – In national cultures, such as that in the UK, the humanities are subject to similar OA requirements as the social sciences, involving monographs being excluded from OA mandates and embargo periods of 12 or 24 months for the archiving of journal articles after publication in a subscription-journal. A few research foundations, such as the Wellcome Trust, will pay for Gold OA to monographs in the medical humanities. It appears likely, given recent moves among European funders, that policies around lengthened embargo periods for the humanities will be harmonized with other disciplines down to zero in coming years.\n\nThe transition to OA of legal literature can be said to be still in its infancy. Legal studies feature some of the lowest OA prevalence levels. In part, this is because of low levels of awareness and little demand for OA publishing outlets amongst legal scholars and practicing lawyers. Those who would most benefit from the OA movement (e.g. law schools unable to subscribe to a wide range of law journals and practitioners in smaller law firms) have little influence over publication behavior. Further, despite the rising importance of international law, the relevance of national legal systems remains high, causing most law journals and law books to focus on the legal situation in a specific country and to be managed by publishing houses in that same country. Often, legal scholars know their publisher(s) personally and tend to publish in a relatively small number of journals -– most of which are closed access.\n\nA) Author behaviour and attitudes – Generally speaking, legal scholars have been reluctant to adopt OA despite agreeing that the research field would benefit from journals that publish OA articles84–86. Even though the field is slowly moving towards OA, many authors of legal publications either are not aware of OA or have little to no incentive to publish their research in OA journals or public repositories87. In legal studies, it is common practice that academics and practicing lawyers publish in the same legal journals or legal commentaries. Some practicing lawyers might even prefer to publish in law journals behind paywalls, thereby guaranteeing an exclusive access to their knowledge and ensuring that potential clients are not able to find the relevant information themselves88. Because of the high relevance of national legal systems, large parts of the legal literature is written in the languages of these countries and published in law journals or books operated in the same countries. Accordingly, the argument that OA enables a worldwide readership is of limited relevance in the field of law. On the other hand, many legal issues are of interest not only to academics and practicing lawyers, but also to the media and politics. According to Hunter (2005), scholarship in law “is arguably the most useful to the public and that has the greatest effect on public policy”. The role of electronic media in supporting scholarly communication and dissemination of research findings is growing but the most important databases (e.g. HeinOnline and LexisNexis in the United States or BeckOnline in Germany) are paywalled89.\n\nB) Publisher behaviour and policies – In the U.S., many or most law reviews are published by law schools, not by for-profit publishers89–91. In contrast to commercial publishers, law schools do not have the usual incentives to oppose OA. Hence, a large and growing number of US law journals are OA. The situation is very different in jurisdictions outside the US where legal scholarship is generally published by commercial publishers84,91. Due to the small demand for OA publishing on part of legal scholars, there are little to no incentives for for-profit publishers to set up new OA journals or book series or to convert existing subscription-based journals to OA. There are some notable exceptions, however. In recent years, some OA law journals have been set up that are predominantly community-driven and operated independently from commercial publishers (e.g. JIPITEC in the EU, Forum Historiae Iuris in Germany or sui generis in Switzerland). According to the DOAJ, there are about 200 OA law journals. OA law journals from the US are in large part not listed, although it is not clear why this is the case. The Creative Commons list of OA Law Adopting Journals lists 37 OA law journals but most of the 18 Harvard Law School Journals (all but one of them are OA) are missing92.\n\nC) Infrastructure of scholarly communication – Most OA journals and open repositories are operated by universities and their law departments. Most universities in the U.S. have their own repositories and also publish their own legal OA working paper series. This idea gains some traction in other countries, for example in Germany, the Netherlands or Italy. Prominent examples of universityled OA journals involve Stanford Technology Law Review, Harvard Human Rights Journal, Bucerius Law Journal or the International Journal of Communications Law & Policy. There is only a limited number of disciplinary repositories and the uptake of repositories such as LawArXiv appears to be slow. In the US and in international law, the most popular disciplinary repository for law professors is SSRN, which is now owned by Elsevier. In English-speaking legal scholarship, scholars find it even difficult to build reputation without being represented in SSRN93. A growing number of universities is further providing support for setting up OA journals or transforming closed to OA journals (for example, by providing an OJS infrastructure). Since practicing lawyers and legal scholars work almost exclusively with texts, OA infrastructures do not have to fulfill demanding technical requirements.\n\nD) Structural and institutional factors – There are three types of work products in legal research: monographs, journal articles and commentaries covering a specific law. PhD theses in the field of law are predominantly published as monographs. Many universities routinely make PhD theses OA (for example Harvard University in the U.S. University of St.Gallen in Switzerland). While the authors of legal books are mostly academics, this remains different for journal articles and legal commentaries where both academics and practitioners contribute. As a result, not only scholars and universities, but also practicing lawyers need to be convinced to move towards OA. One possible way to foster OA amongst legal scholars might be to encourage academics and practitioners to publish in different journals and commentaries. In this scenario, academics could publish their works in scientific OA journals and practitioners could keep on using closed access journals and commentaries, which, however, would be more practice-oriented. Research project costs often are smaller in the field of law compared to other disciplines. As legal scholars are not dependent on third party funding, so that funder OA requirements have only limited potential to incentivize OA publishing.\n\nE) OA mandates and policies – OA mandates by public funding agencies, research foundations and private companies only have limited impact in the field of law since legal research is relatively inexpensive and therefore does not depend on third party funding in large parts90. As law is often considered as a discipline related to the humanities, scholars in this field face the same OA requirements as the social sciences and humanities, including relatively long embargo periods for Green OA and monographs that are excluded from OA requirements. It can be assumed that OA mandates by universities, if mandatory, will have a greater potential to foster some change in the field of legal academia than OA mandates by public funders. An important alternative to top-down OA mandates are OA policies from law schools (for example the Harvard Law School Open Access Policy) and non-binding statements promoting OA. In 2009, the directors of the law libraries of 12 US Universities signed the Durham Statement on Open Access to Legal Scholarship. This statements urges law schools to make the definitive versions of journals and other scholarship produced at the school immediately available upon publication in stable, open, digital formats, rather than in print94.\n\n\nDiscussion and conclusion\n\nOver the course of the last three decades, OA to the scholarly literature has emerged as a new norm of scholarly publishing. As a response to perceived limitations of the subscription-based model of scholarly publishing and propelled by technical possibilities offered by the Internet, OA promises the removal of major barriers in assessing, distributing and re-using research findings6. OA publishing has grown substantially across different types of publication outlets, academic disciplines and research contexts, resulting in growing shares of scholarly publications being made openly accessible. While there is little doubt about the notion that OA is of global relevance with the potential to revolutionize the ways in which scholarly publications are shared, many of the discussions surrounding OA still revolve around the question of how it affects publishing practices across different academic disciplines. This question has become increasingly relevant against the background of first, funding organisations, governments and universities implementing OA mandates and policies that require scholars across all disciplines to make their research outputs OA and, second, vast amounts of resources being dedicated to the development, maintenance and advancement of respective publishing infrastructures.\n\nReviewing bibliometric studies that assessed OA prevalence and publishing patterns across broad academic disciplines in the first part of this review, we examined how different disciplines have adopted OA publishing over time and identified discipline-specific patterns of OA publishing. In the second part of this review, and based on a social shaping of technology perspective, we examined a variety of data sources and identified discipline-specific barriers and potentials for OA. Doing so, we explained the publishing patterns and trends observed in the first part of this review. We found that, over the last three decades, scholarly publishing has experienced a shift from closed access to OA. The proportion of scholarly literature that is openly accessible has increased continuously across all disciplines, resulting in overall OA levels well above 50% for publication years after 2010. Most OA appears to be published as journal articles in subscription journals for which the accepted of the published version can be retrieved from an open repository (Green OA). Publication of articles in pure OA journals (Gold OA) is also of importance for scholarly publishing, even though the relative uptake on Gold OA remains well below Green OA for most publication years and academic disciplines. Hybrid OA generally is of little variance for OA publishing, with 1% or less of all scholarly outputs being published as articles free under open licenses in subscription journals. The importance of Bronze OA is comparable to Gold OA, featuring similar levels of uptake. Having compared OA publishing patterns for the broad academic disciplines natural and technical sciences, medical sciences, social sciences, law and humanities, we found that the shift of scholarly publishing towards OA occurs uneven across disciplines in two respects. First, the growth of OA has not been uniform across disciplines; scholars in different disciplines differ substantially in how much they embrace the idea of OA, which manifests itself in varying proportions of openly accessible research outputs across disciplines and sub-disciplines. Second, academic disciplines have not converged on a set of homogeneous OA publishing practices, but differ substantially regarding the OA publishing channels scholars use to publish their research outputs OA. This unfolds as follows: scholars in medical and health-related sciences initially were reluctant to adopt OA publishing, but soon the OA uptake in these disciplines increased substantially and particularly biomedicine and clinical medicine took on leading roles in embracing OA. Medical scholars make research outputs openly accessible predominantly by publishing them in journals: The Gold OA route is of central importance for OA in medical sciences, followed by Hybrid, Bronze, and, with some distance, Green OA. Factors facilitating OA and shaping OA publishing practices in these disciplines are strong OA mandates combined with both funder-operated repositories and available funding for APCs, a richness in highquality OA journals and the perception of authors that OA journals allow for a wider circulation of publications than subscription journals do. The medical sciences are closely followed by the natural and technical sciences in embracing the idea of OA. Within this broad discipline, however, we found different patterns of OA publishing both in terms of OA prevalence rates and OA routes: Scholars in physics, mathematics, information technology, astronomy and biology were the early pioneers of OA and continue to make large shares of their research outputs OA, whereas scholars in engineering and chemistry are more reluctant to make research outputs openly accessible. Further, while Green OA plays an important role for scholars in physics, mathematics, information technology, astronomy and biology (followed by Bronze, Gold, and with some distance, Hybrid OA), scholars in engineering and chemistry publish most OA through the Gold OA route. OA in physics, mathematics, information technology, astronomy and biology has been facilitated by an existing culture of preprint distribution and by high levels of familiarity with OA publishing in general and Green OA in particular. Barriers to OA in chemistry and engineering can be identified as concerns about the quality of OA journals, which are shared by scholars, publishers and learned societies alike, as well as high degrees of industrial integration within these fields. The OA uptake in the social sciences is well below the medical and natural and technical sciences, but remains above OA prevalence rates that we observed for the humanities and law. For scholars within the social sciences, open repositories appear to be of central importance for making research outputs openly accessible, closely followed by publication in Gold OA journals, and, with some distance, Hybrid and Bronze OA. We identified several factors that shape OA publishing practices within the social sciences. Most importantly, this includes authors’ concerns about the quality and prestige of OA journals, the central role of monographs in terms of academic career advancement and difficulties in assessing funding for APCs and BPCs. These factors also explain why most OA within the social sciences is published via the Green route. We observed signs of cultural change particularly in young scholars, who embrace the idea of conducting science more openly. Humanities features OA uptake levels well below the social sciences. Most OA within the humanities is published as Hybrid OA, followed by Green OA, Bronze OA and Gold OA. The most important factors shaping these publishing practices are comparable to those identified in the social sciences, including a dry climate for APC and BPC funding, the central role of monographs, which are less likely to become OA, and authors, publishers and scholarly societies being opposed to OA. Just like in the social sciences, there is, however, some movement with new economic models that do not rely on author payments appearing to have some traction with humanities scholars. OA in law is still in its infancy with legal scholars making only small proportions of their research outputs OA. In large part, this is due to low levels of awareness and little demand for OA within the academic community. Of relevance is also that OA mandates and policies only have limited impact on publishing behaviour as legal studies in large part do not depend on third party funding. The financing of publication fees for publishing in OA journals appears to constitute a major barrier to OA within the humanities, social sciences and law. We believe that new OA models that do not rely on author payments represent a viable alternative to financing OA within these disciplines. This includes models such as the OLH or other crowd funding initiatives, such as KU.\n\nThese findings indicate that, as OA is implemented and used across different academic disciplines, it is shaped by the scholars that use respective communication technologies. In turn, OA technologies shape the ways in which scholars communicate and disseminate their research findings. Our findings also suggest that, in spite of the transformational potential of OA, the shift towards OA is uneven across disciplines and even sub-disciplines. We found that academic disciplines feature distinctive research cultures that have grown historically and manifest themselves in discipline-specific publishing practices. These publishing practices vary fundamentally in terms of their compatibility with OA publishing formats, which is the reason why the implementation of OA can be assumed to be a natural continuation of publishing cultures in some disciplines, while in other disciplines, the implementation of OA faces major obstacles and requires a change of research culture.\n\nOur review has several limitations and these should be taken into account when interpreting our results. First, most of the bibliometric studies included in our review assessed OA publishing practices across broad academic disciplines, that is, the natural and technical sciences, medical sciences, social sciences, humanities and law. Choosing broad academic disciplines as units of analysis produces data that is fairly coarse-grained. Consequently, there is a chance that relevant differences in publishing practices between sub-disciplines remain undetected. For example, the few bibliometric studies that have assessed OA publishing practices for the natural sciences and related sub-disciplines revealed that there are substantial differences in the OA uptake between physics and chemistry. Therefore, we encourage future bibliometric research to assess OA publishing practices not only across broad disciplines, but to also take into account related sub-disciplines and research fields. Second, only two bibliometric studies in our review have included Bronze OA and Hybrid OA in their analyses, resulting in highly limited data on the relative uptake on these OA routes. This likely limits the robustness of our conclusions. We encourage further research to include Bronze and Hybrid OA in their bibliometric analyses. Third, in explaining OA publishing patterns, we conducted a narrative review by the means of which each co-author identified relevant socio-technical forces that affect OA within their area of research training. A major limitation of narrative reviews is that there is a chance that evidence has been selectively chosen. We tried to keep limitations in objectiveness to a minimum by basing the narrative review on an analytical framework.\n\nOverall, our review is the first to comprehensively explain OA publishing patterns across academic disciplines. We identified patterns and trends of discipline-specific OA publishing practices and revealed barriers and potentials for OA across disciplines. Doing so, we contributed to understanding how different disciplines adopt and shape OA. We encourage further research to investigate the underlying mechanisms and factors that shape scholarly communication in general and OA publishing practices in particular. A profound understanding should inform both OA policies and community-driven efforts in promoting OA.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nPreliminary findings of this study were presented at the 12th Open-Access-Tage: Varieties of Open Access conference, Graz, Austria from the 24th–26th September 2018. Severin, A. (2018, October). Der Weg zu Open Access in den Geistes- und Sozialwissenschaften: Publikationspraktiken, Hindernisse und Potentiale. A copy of the presentation is available in German on Zenodo: http://doi.org/10.5281/zenodo.1441034.\n\nThe authors would like to thank Peter Suber for valuable comments to the Law chapter. We would also like to thank Alexander Grossmann for valuable comments on the terminology “Bronze OA”.\n\n\nFootnotes\n\n1 It has been discussed controversially whether or not this type of publication is is in fact OA. A case has been made that such publications are not (Bronze) OA, but “free-to-read” only13, whereas others argue that Bronze OA is a sub-category of OA as OA is not a binary category, but encompasses a range of components that determine the degree of openness of a certain publication outlet14. Following the latter argument, we use the term Bronze OA throughout this publication and acknowledge the fact that there are varying degrees of openness.\n\n2 For a comprehensive discussion of the merits of these perspectives in explaining publishing practices see Kling & Kim (2000) and Oostveen (2004).\n\n\nReferences\n\nBjörk BC, Solomon D: Open access versus subscription journals: a comparison of scientific impact. BMC Med. 2012; 10(1): 73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLamb C: Open access publishing models: opportunity or threat to scholarly and academic publishers? Learn Publ. 2004; 17(2): 143–150. Publisher Full Text\n\nHagner M: Open Access, data capitalism and academic publishing. Swiss Med Wkly. 2018; 148(78): w14600. PubMed Abstract | Publisher Full Text\n\nGinsparg P: It was twenty years ago today.... 2012; 21(1). Publisher Full Text\n\nKling R, Mckim G: Not Just a Matter of Time: Field Differences and the Shaping of Electronic Media in Supporting Scientific Communication. J Am Soc Inf Sci. 2000; 51(14): 1306–1320. Publisher Full Text\n\nTennant JP, Waldner F, Jacques DC, et al.: The academic, economic and societal impacts of Open Access: an evidence-based review [version 3; referees: 4 approved, 1 approved with reservations]. F1000Res. 2016; 5: 632. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarnad S, Brody T: Comparing the Impact of Open Access (OA) vs. Non-OA Articles in the Same Journals. D-Lib Magazine. 2004; 10(6). Publisher Full Text\n\nOdlyzko AM: Tragic loss or good riddance? the impending demise of traditional scholarly journals. 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Reference Source\n\nMacKenzie D, Wajcman J: The Social Shaping of Technology. Open University Press, Birmingham, UK, 2 edition, 1999. Reference Source\n\nWilliams R, Edge D: The social shaping of technology. Res Policy. 1996; 25(6): 865–899. Publisher Full Text\n\nCawley A, Hynes D: Evolving mobile communication practices of Irish teenagers. Aslib Proc. 2010; 62(1): 29–45. Publisher Full Text\n\nKamete AY: A Concept ‘Vandalised’: Seeing and Doing e-Planning in Practice. International Journal of E-Planning Research. 2018; 7(1): 1–14. Publisher Full Text\n\nKenney R, Warden R: An Open Access future? Report from the eurocancercoms project. Ecancermedicalscience. 2011; 5: 223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchroter S, Tite L, Smith R: Perceptions of open access publishing: interviews with journal authors. BMJ. 2005; 330(7494): 756. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWarlick SE, Vaughan K: Factors influencing publication choice: why faculty choose open access. Biomed Digit Libr. 2007; 4(1): 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrass W, Cross J, Gardner V: Taylor & Francis Open Access Survey June 2014. Technical Report June, Taylor & Francis / Routledge, 2014. Reference Source\n\nRowley J, Johnson F, Sbaffi L, et al.: Academics’ behaviours and attitudes towards open access publishing in scholarly journals. J Assoc Inf Sci Technol. 2017; 68(5): 1201–1211. Publisher Full Text\n\nLwoga ET, Questier F: Open access behaviours and perceptions of health sciences faculty and roles of information professionals. Health Info Libr J. 2015; 32(1): 37–49. PubMed Abstract | Publisher Full Text\n\nSánchez-Tarragó N, Carlos Fernández-Molina J: The open access movement and Cuban health research work: an author survey. Health Info Libr J. 2010; 27(1): 66–74. PubMed Abstract | Publisher Full Text\n\nSandesh N, Wahrekar S: Choosing the scientific journal for publishing research work: perceptions of medical and dental researchers. Clujul Med. 2017; 90(2): 196–202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoher D, Shamseer L, Cobey KD, et al.: Stop this waste of people, animals and money. Nature. 2017; 549(7670): 23–25. PubMed Abstract | Publisher Full Text\n\nHawley JB: The jci’s commitment to excellence — and free access. J Clin Invest. 2003; 112(7): 968–969. Publisher Full Text | Free Full Text\n\nSchroter S: Importance of free access to research articles on decision to submit to the BMJ: survey of authors. BMJ. 2006; 332(7538): 394–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler D: Wellcome Trust launches open-access publishing venture. Nature. 2016. Publisher Full Text\n\nButler D: Gates Foundation announces open-access publishing venture. Nature. 2017; 543(7647): 599. 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PubMed Abstract | Publisher Full Text | Free 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\nEndres K, Spiecker C: Open Access to Scientific Information: Engineering. Reference Source\n\nVan Noorden R: Particle-physics papers set free. Assessed 02.11.2018. Reference Source\n\nOpen Access Policy for CERN Publications. Technical Report CERN-OPEN-2017-020. CERN, Geneva, 2017. Reference Source\n\nRodriguez JE: Awareness and Attitudes about Open Access Publishing: A Glance at Generational Differences. J Acad Libr. 2014; 40(6): 604–610. Publisher Full Text\n\nWijewickrema M, Petras V: Journal selection criteria in an open access environment: A comparison between the medicine and social sciences. Learn Publ. 2017; 30(4): 289–300. 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}
|
[
{
"id": "42144",
"date": "05 Feb 2019",
"name": "Richard Smith",
"expertise": [
"Reviewer Expertise Peer review",
"journalology",
"NCD",
"research misconduct. global 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\nI’m sorry that I haven’t been slow with reviewing this study, but the increasing length of studies, which generally I think necessary, makes peer review even more onerous.\nImportance:\nThe study asks two important questions - what is the variation in use of open access by discipline, and what might be the reasons for the differences? And it provides answers that are generally convincing, although hard to digest.\n\nOriginality:\nIndividual studies provide some answers to these questions, but this review brings them together. That is useful.\n\nMethods and presentation:\nAlthough the study is described as a meta-synthesis, it seems to me that it’s essentially a descriptive study. The authors find the studies and then largely describe what they report. There isn’t much synthesis, and I, as a reader, would appreciate more.\n\nThere is huge heterogeneity in the studies of proportions of articles that are open access, but readers are left to look through a table that is four pages long - and largely to wonder for themselves about the reasons for the big variations. I know that all the data are there, but how is it that a study of 12m article from 2018 finds that 66% of articles are open access, whereas another equally large study from the same year finds that 29% are open access? Clearly they must be using different definitions of open access, and the authors don’t themselves define open access or its subsets (gold, green, hybrid, and bronze). They should. I’d like to see a summary table that has the following columns: Year of study, number of articles in the study, definition of open access, proportion of articles that were open access.\n\nThere should be another summary table that shows the variation by discipline in overall open access rates and in the subsets of open access.\n\nAnother useful addition would be a figure that showed how proportions of articles that are open access by discipline have varied over time.\n\nThe authors might also give a table that shows the association between their “analytical dimension” and the different disciplines. At the moment readers can get this information only by ploughing through pages of text that describe the variations by discipline.\n\nAs this is essentially a descriptive study I don’t have great criticisms of the methods and conclusions, although I recognise the weakness of surveys, which give us data on what people say about their behaviour than what really drives their behaviour.\n\nMy biggest criticisms are about presentation. I’d like to see not only more tables and a figure but also a much shorter paper backed up by supplementary material if necessary. That would, I judge, make for a much more readable and useful paper and dramatically increase the number of readers.\n\nMinor points:\nThere is at the moment a great deal of repetition. The discussion is mostly a repetition of the results.\n\nI’d like to see some data in the abstract.\n\nI believe that abbreviations are a plague in medical writing, and I’d avoid them, including OA for open access.\n\nI’d drop “evidence-based” in the title.\n\nIt would be good to have the search strategy as a table.\n\nMuch of what’s in the results should ideally be in the discussion.\n\nThere are a fair few typos.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5337",
"date": "16 Apr 2020",
"name": "Anna Severin",
"role": "Author Response",
"response": "We thank Richard Smith for his helpful review of our manuscript. In response to his comments, we have made revisions throughout version 2: Methods and presentation: In an attempt to restructure the paper, we have included more synthesis and less description of results. We now discuss the heterogeneity of the studies included in our review (in terms of their methodological approaches and results) throughout the paper (please see Methods, Results and Discussion). We have added a sub-section in which we state our definition of OA and its routes (please see Methods and Table 1.) In Table 2, we have added a column on the OA definition of each study included in our review. Throughout the Results and the Discussion section, we note if included bibliometric studies differ from our definition of OA. Unfortunately, F1000 does not support supplementary files / appendices and requires all results to be included as part of the main text of the paper. This means that Table 3 (now Table 4) could not be moved to the supplementary files. In order to keep the paper short, we therefore decided not to add any further tables or images. Information on year of study, number of articles in the study, definition of open access is given in Table 2. Information on proportions of articles that were open access and in the subsets of open access is included in Table 3. Where appropriate, we discuss how the methodological heterogeneity of the studies included in our review affects the consistency and comparability of their results (please see Methods, Results and Discussion). We restructured and shortened the text significantly. We now illustrate the analytical dimensions (i.e. the different phases / eras of OA development) in a clear structure, albeit without a separate table. We now acknowledge the weakness of author surveys as a limitation (please see Discussion). We removed repetitions, including the repition of results in the discussion. We added data to the abstract. To keep the paper short, we decided to keep the abbreviations, including OA for open access. We dropped “evidence-based” in the title. We added the search strategy as a figure (please see Figure 1). We moved parts of the results to the discussion and removed repetitions in both chapters. We corrected all typos."
}
]
},
{
"id": "43517",
"date": "12 Feb 2019",
"name": "Mikael Laakso",
"expertise": [
"Reviewer Expertise scholarly publishing",
"open access",
"information systems 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 manuscript tackles a very important and ambitious topic, that of improving our knowledge about what differences there are in open access uptake across academic disciplines. A lot of bibliometric work has been done in this area, however, most of it has been fragmented as definitions and methodological approaches have varied a lot across studies. This study makes a welcome exception to most of the research within the field in not just producing yet another measurement of OA that is improved in some incremental way, yet failing to be compatible with results of earlier studies, but rather leverages what is already out there (both in terms of existing studies, but also other knowledge) in order to thoroughly discern how disciplines differ in their approaches to utilising various forms of OA. The manuscript has two main components 1) a systematic literature review of bibliometric research (which includes 11 articles), and 2) an analysis of open access in academic research disciplines interpreted through the theoretical lens of Social Shaping of Technology. I could easily see both parts being published as individual articles based on what they aim to achieve and in how challenging they are to put together, having them together like this is not a major problem but something that requires effort and rigour which this first version of the manuscript succeeds with to a satisfactory degree. The text itself is of high quality.\n\nIs the work clearly and accurately presented and does it cite the current literature?\n\nFor the most part, yes, but I do think the strictness of the criteria for the systemic literature review of OA uptake requires that supplementing research that is left out of the review is still discussed/reflected upon as in some other parts of the manuscript. I have a couple of recommendations for this that are mentioned below in this section.\n\nOne source which I think is a great omission and gives a lot of detailed breakdown into the differences between OA journal differences is: Crawford, W. (2018). GOAJ3: Gold Open Access Journals 2012-2017. https://walt.lishost.org/2018/05/goaj3-gold-open-access-journals-2012-2017/. If “top-down” studies, focusing on only one type of OA mechanism, were excluded this study was perhaps not included on such grounds but I think it is doing the study a disservice – there is no better source that describes the disciplinary differences longitudinally across disciplines, including information about article processing charges, than that e-book and associated dataset. If not integrated into the meta-analysis it should at least be used in the other parts of the manuscript to frame the study and its results.\n\nFurther reference you could consider, purely based on the idea that they have also explored disciplinary differences in the OA context specifically, albeit through analysis of bibliographic indexes:\n\nLiu, W. and Li, Y. (2018), Open access publications in sciences and social sciences: A comparative analysis. Learned Publishing, 31: 107-119. doi:10.1002/leap.11141\n\nEnnas, G. and Diguardo, M.C. (2015), “Features of top-rated gold open access journals: an analysis of the Scopus database”, Journal of Informetrics, Vol. 9 No. 1, pp. 79-892.\n\nGadd, E., & Covey, D. T. (2019). What does ‘green’ open access mean? Tracking twelve years of changes to journal publisher self-archiving policies. Journal of Librarianship and Information Science, 51(1), 106–1223\n\nIn general I avoid suggesting citing material that I have been involved in authoring as part of reviews I have conducted, but in this case I would like to point out two studies that give precise metrics of various types of OA in narrowly defined disciplines, and another study which deals with disciplinary differences in self-archiving rights, utilizing these references is completely optional and not something that influences my verdict or recommendation for a revised version of the paper:\n\nLaakso, M. & Polonioli, A. Scientometrics (2018) 116: 2914.\n\nLaakso, M., & Lindman, J. (2016). Journal copyright restrictions and actual open access availability: A study of articles published in eight top information systems journals (2010–2014). Scientometrics, 109(2), 1167–11895.\n\nLaakso, M. (2014), “Green open access policies of scholarly journal publishers: a study of what, when, and where self-archiving is allowed”, Scientometrics, Vol. 99 No. 2, pp. 475-4926.\n\nTable 3, being split onto 4 pages, is massive and very hard to use for making any conclusions between time/discipline(which each study having their own way of classifying as well)/OA method by eye. It is functional but far from optimal. In this case I would save a table like this to become an appendix, and rather compose a figure where the discipline categories have been standardized according to some well established scheme that fits well with most of the studies. This would come at the cost of precision in losing sub-discipline breakdowns in many cases but in my view that is worth the cost.\n\nI would also suggest to focus less on comparisons of decimal point-accuracy prevalence of OA mechanisms between the previous studies, since they vary so much depending on other factors than inherent disciplinary differences. Zooming out would make it easier to see, and tell the reader, what is important to focus on, not just drop the decimal points but also consider putting in subheadings or structuring the “Prevalence and patterns of open access publishing practices: Meta-synthesis of bibliometric studies” so that each “era” of OA development would get its own mini-narrative, now its just a long single block of text and a lot of percentages that are hard to relate to anything.\n\nThe influence of academic social networks is in my view underrepresented in the review of existing literature and conclusions of the study, they have provided a substantial share of the OA copies measured in the various bibliometric studies and many authors also perceive them as essentially “solving” the issue of OA and paywalls on a personal level since there has been very weak monitoring of adherence to copyright on such services.\n\nThe concept of Bronze OA would need further unpacking since in most of the reviewed studies it is present, but not always separated and referred to as such from other OA provision mechanisms.\n\nIs the study design appropriate and is the work technically sound?\n\nThe collection process for inclusion of existing literature contains both strict elements (specific indexes were queried with specific identical keywords, studies had to fulfil four pre-set criteria to be included) but also what seems like a liberal and flexible amount of bottom-up/explorative elements (authors contributing discipline specific OA findings/literature, scouring reference lists and Google Scholar profiles). This large jump between very strict and transparent, to a largely undocumented part where “anything goes” which has very little transparency other than the disciplinary analysis´ themselves could be expanded somewhat.\n\nFor me it was a bit unclear what the first criteria in Table 1 when strictly applied entails, do the studies have to explore OA availability “bottom-up” through web-search engines/querying and giving uptake metrics for various OA mechanisms in one single study? If this is the case, which it could be by looking at the included studies, the criteria description should in my view be revised to communicate this.\n\nThe time-lag between when a study has measured the level of OA and when the materials being measured were published, varies a lot across the included studies. I think this caveat/feature could be highlighted more in the text because it matters quite a lot if an article was searched for 1 year after it was published or 5 years after it was published.\n\nAre sufficient details of methods and analysis provided to allow replication by others?\n\nYes, the study is literature-based with no need for further data.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\n\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility?\n\nYes, the study is literature-based with no need for further data.\n\nAre the conclusions drawn adequately supported by the results?\n\nYes, my revision suggestions concern mainly minor points not critical to the main results and contribution of the study.\n\nThe second half of the manuscript, which comprises the discipline-specific description of OA practices, I have very little to comment about since I think it does a great job at mixing research results with discipline-specific knowledge. The most central things are brought up and argued for well.\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": "5338",
"date": "16 Apr 2020",
"name": "Anna Severin",
"role": "Author Response",
"response": "We would like to thank Mikael Laakso for his helpful comments. In response to his review, we have made the following revisions throughout the manuscript: We agree that the strictness of inclusion criteria for the systematic review might have caused studies to be left out that also analysed disciplinary OA publishing practices, albeit from a different perspective. We acknowledge this limitation in the Discussion. To frame our results, we now include disciplinary studies in the narrative review and in the Discussion section. This includes some of the references you provided us with – thank you. We agree that Table 3 (now Table 4) is large and we would have liked to move it to the appendix too. Unfortunately, F1000 no longer supports supplementary files / appendices and requires all results to be kept as part of the main text. We therefore kept Table 3 (now Table 4) as is. To keep the paper short, we did not add any further figures or tables. We restructured and shortened the text significantly. We now focus less on comparisons of decimal point OA prevalence levels and only highlight the most significant figures. We further restructured the section “Prevalence and patterns of open access publishing practices: Meta-synthesis of bibliometric studies” and included more subheadings. We now illustrate the different phases / eras of OA development in a clearer structure, albeit without sub-headings for the different eras (as we believed this to be too fine-grained). We now discuss the importance of social networks and so-called “free availability” for making scholarly publications openly accessible (please see Methods, Results and Discussion section). We now define Bronze OA and discuss how it is defined and measured by the studies included in our review (please see Table 1, Table 3, Results and Discussion section). To address the fact that the collection process included both strict alements and a more liberal amount of explorative elements, we have added information on how we conducted the narrative review of discipline-specific OA literature (please see Methods). Further, we now state that the strictness of inclusion criteria for the systematic review might have caused studies to be left out that also analyzed disciplinary OA publishing practices. We also state that, as for our narrative review, there is a chance that evidence has been selectively chosen (please see Discussion) Yes, studies had to explore OA availability “bottom-up” through web-search engines/querying and giving uptake metrics for various OA mechanisms. We have added this information as a criterion for inclusion in our review (please see Table 1 and Methods). The discussion now includes the time-lag between when a study has measured the level of OA and when the materials being measured were published as a limitation to our study."
}
]
},
{
"id": "43518",
"date": "19 Feb 2019",
"name": "Birgit Schmidt",
"expertise": [
"Reviewer Expertise library and information science",
"scholarly communication",
"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\nThe article reviews and synthesises quantitative and qualitative findings on disciplinary OA publication practices and uptake over time. The article extensively reports figures on OA levels from former studies (selected based on a set of criteria) and discusses factors influencing disciplinary publishing cultures based on a framework of analytical dimensions.\n\nOverall, this creates a useful comprehensive overview and reference point.\n\nIs the work clearly and accurately presented and does it cite the current literature? A few references should be reconsidered, e.g. on p. 11 the authors cite Kling and Kim (2000) for a statement that “social and cultural factors are believed to be of less or no importance in explaining the emergence of OA”. This does not seem justified as Kling and Kim’s study adopts a social shaping perspective, in order to analyse field differences in the use of electronic media. Some references are certainly not the most authoritative, e.g. websites that summarise disciplinary attitudes (e.g. reference 52). For further comments on references see the list below.\n\nIs the study design appropriate and is the work technically sound? The authors search and selection criteria for studies on the uptake of OA are sound. However, in some cases that leads to limitations as several studies, in particular the earlier ones, only provide little information about the status for certain disciplines, e.g. the humanities. In such cases it would have been good to further amend the data by taking into account disciplinary studies. The authors note that there is little consistency in the reported uptake of OA, however, this should be discussed earlier and in more detail in the study.\n\nAre sufficient details of methods and analysis provided to allow replication by others? The description of methods on how studies were retrieved and selected as well as the framework of the analysis seem sufficient. However, too little attention is given to major differences of the considered studies, in particular the definitions of OA used, the data sources (including how was OA identified), and how the limitations of the studies should be considered in the comparison of study results.\n\nThe authors’ definition of the different OA routes is certainly not homogeneously applied by all selected studies, as they mainly follow Piwowar et al.’s (2018)1 approach to define OA as exclusive categories. More commonly, “green OA” is defined as “self-archiving” of peer-reviewed works in OA repositories, i.e. green OA overlaps with other OA routes.\n\nAnother deviation is e.g. the study by Archambault et al.(2014) (reference 10) which considers green OA as “OA provided before or immediately after publication by author self-archiving” while focusing on deposit in “institutional repositories and some thematic repositories listed in OpenDOAR and ROAR” (Ibid., pp. 4 and i), e.g. arXiv.org was included; PubMed Central was considered under Other OA. In particular, it can thus be expected that in this case preprints have been included.\n\nThere are also a number of results in some of these studies which deserve further attention – and if possible explanation – in particular, if these have not been observed in other studies: e.g. the high rate of hybrid OA for Mathematics and the Humanities reported by Piwowar et al.(2018)1. Another example are the surprisingly high gold OA figures found by Jamali and Nabavi (2015)2, please make explicit how gold OA was defined.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nThere is no statistical analysis across the selected studies, the authors mainly provide an overview of the reported levels of OA, and organise these in three phases. In general, the authors report all available figures of OA uptake by discipline but do not provide any comments on the sometimes vast differences of these figures.\n\nAre all the source data underlying the results available to ensure full reproducibility? The authors have created summary tables of relevant studies and their framework for the further analysis. Regarding the uptake of OA the authors refer to data in the selected studies but have not created additional comparative data. All additional information which was used in the narrative review of mechanisms and factors shaping OA publishing is disclosed through the list of references. Some of the considered studies are not reproducible based on the chosen methodology (e.g. often a web search for an openly available version of a research article was applied, which may change over time).\n\nAre the conclusions drawn adequately supported by the results? Yes, overall the main conclusions are adequately supported. There are in addition a couple of other issues and small errors which I would like to point out:\nTypos: “66& for publication years…” - use %; replace “SEM” by “STM” for “Science, Technology and Medicine”; replace “PLoS” by “PLOS”. p. 4: The conclusion regarding the comparison of OA levels for medical research areas and the humanities is not quite convincing as only one figure for the humanities (based on Gargouri et al., 2012 (see reference 9)) is provided. Table 2: Consider adding more information on the definition of OA or a specific column which explains how the study deviates from the definition of OA provided by the authors. It is also important to note which studies treat the definitions as exclusive categories and which used definitions of OA that allow overlaps, in particular with green OA. Add “with DOIs” in column two of Piwowar et al (2018). The reference European Commission (2014) should be cited as Archambault et al. (2014). Table 3: The second column on Piwowar et al. (2018) should include a note that the figures for the Humanities and the Arts were not included as these disciplines are underrepresented in the WoS and in terms of DOI coverage. In the Hybrid OA column the overall figure cannot be correct: 0.6% is too low; according to Table 3 of Piwowar et al. it should be replaced by 4.3%. Even if certain routes of OA have not been assessed, e.g. Hybrid and Bronze OA, it would be good to add a note if they have been included in other categories. Please check the figures provided for Martín-Martín et al. (2018)4, the overall OA figures do not seem to match with the sum of the OA routes (the sum is substantially smaller, what is missing? E.g. Social and Behavioral Sciences: 49.9% OA vs. a sum of 23.4% for all OA routes). For further studies the overall OA figures do not match with the sum of the OA routes either, e.g. for Science Metrix (2018)4 and Jamali and Navabi (2015)5, please explain. p. 10: Please correct the underestimation of Hybrid OA, as noted above the overall figure found by Piwowar et al.(2018)1 is 4.3% for recent articles with a DOI in WoS. p. 11: The statement “… as the OA model is unlikely to generate the level of income and profit that can be achieved with the subscription model” deserves a reference. p. 11: “This also applies to academic and professional societies” – the given reference does not seem to be connected to this statement. Table 4: “APC levels” is a bit narrow as a perspective, and not much is said about cost aspects in the later discussion. Regarding infrastructures the support aspect is missing (e.g. institutional support for the green and gold OA routes). Regarding structural and institutional factors copyright is missing (this is not just an aspect which is relevant for publishers).\n\np. 12: NIH OA mandate – mention the year in which the mandate was introduced p. 13 E) does not mention Wellcome Open Research p. 13 A) Preprints do have a tradition in biology, but were mainly circulated in small circles of colleagues. Please note that on arXiv.org quantitative biology represents a very small share and is not representative for the discipline. A reference for molecular biology is e.g. the study of Kling and Kim (2000)6. Thus the conclusions on green OA do not fully apply to biology.\n\np. 14 C) You provide several examples of OA journals in Chemistry, what does “enjoy popularity” mean here? p. 14 D) Please provide a reference for the stated national focus of engineering. p. 14 E) Remove bold face for the word “Reflecting”. p. 14: There seems to be some redundancy in the introduction to “Open access in the social sciences”. Moreover, please add a reference for the order of OA routes as the one you state is not found by the most recent studies (compare e.g. Piwowar et al1.). p. 14 A) Reference 51 seems a bit old for a statement about the present state. p. 15 – typo “Open library of Humanities” should be “… Library …” p. 15: “While long-term access to research outputs is questionable in these models…” – this is an unjustified statement. p. 15 C) You do not provide any evidence for growth of the number of repositories when you state the current status, please justify this statement. p. 15 C) “Because OA preprint repositories do not employ peer review, however, social scientists have been slow to adopt Green OA.” – This statement is not convincing, preprint servers circulate non-peer reviewed versions while green OA focuses on final author manuscripts (after peer review) or the publisher’s version (i.e. you compare apples and pears here). p. 15 C) Some of the details about academic journals could be moved to B). p. 16 OA in the humanities: Add reference to the cited order of green OA, Bronze OA etc. p. 16 A) The last sentence should be amended, otherwise it is just a cliché pointed out but not put to rest. p. 16 C) MLA Commons is a network of scholars, the related repository is called CORE. p. 16 C) You state that “post-publication peer review remains rare … with a few notable notable exceptions and experiments”. – Please note that one of your references do not support this statement: Bourke-Waite (reference 83) does not elaborate on post-publication peer review but points out that for the HSS double-blind peer review remains the norm and notes an experiments of open peer for monographs by Palgrave-Macmillan. Brienza (2012)7 certainly fits here as for the book of K. Fitzpatrick post-publication peer review was applied to a draft version (in the sense of open peer commenting). It could be argued that review articles which are quite common for monographs to be considered as a form of post-publication peer review. p. 16 E) For the last sentence you may add an indicative reference, e.g. to Plan S which does not allow any embargoes. p. 17 OA in law: Please add references to the statement that legal studies feature some of the lowest OA levels. p. 17 B) JIPITEC is based in Germany (which is in the EU…). You note that several journals are not listed in the DOAJ which is indeed a pity, they may not have re-registered after the introduction of revised criteria in 2014. p. 18 “overall OA levels well above 50%” – add reference, not all recent studies agree on this point; “the relative uptake on Gold OA remains well below Green OA for most publication years and disciplines” – add reference; “hybrid OA generally is of little variance” – not according to Piwowar et al. (2018); “hybrid OA… with 1% or less of all scholarly outputs” – this figure is too low, Piwowar et al. (2018) found 4.3% hybrid OA on average p. 18 “… biology were the early pioneers” – see comment on biology above. OA in biology is not initially strong via preprints and green OA (arXiv.org only holds a limited number of quantitative biology papers), uptake gets stronger via gold OA with the wave of new OA journals in the 2000s, and only from 2013 onwards preprints finally take off (launch of PeerJ Preprints and bioRxiv). For a discussion of earlier initiatives/experiments with biology preprints which were pretty much blocked by the publishing industry see: Cobb, M. (2017). The prehistory of biology preprints: A forgotten experiment from the 1960s8. p. 18 “Barriers to OA in chemistry and engineering can be identified as concerns about the quality of OA journals” – add reference p. 19 “difficulties in assessing funding for APCs and BPCs” – you mean “accessing” here, please also add a reference. p. 19 “we observed signs of cultural change particular in young scholars” – add reference p. 19 “Most OA within the humanities is published as Hybrid OA…” – try to explain why hybrid OA comes first. Piwowar et al. (2018) which seems to be the source here do not seem to do. p. 19 “This includes models such as OLH or other crowd funding initiatives, such as KU.” – You may consider adding a reference here, e.g. Bulock, C. (2018)9. p. 19 “… OA … is shaped by the scholars that use respective communication technologies.” – You do not really discuss different types of communication technologies but mainly point out common communication formats, channels and infrastructures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5339",
"date": "16 Apr 2020",
"name": "Anna Severin",
"role": "Author Response",
"response": "We would like to thank Birgit Schmidt for her useful peer review of our study. In response to her comments, we have made the following revisions throughout the manuscript: We agree that the reference Kling and Kim (2000) on p.11 should have been reconsidered. Shortening and restructuring of the paper removed the statement from the text altogether. As for references that were not authoritative (e.g. reference 52), we either replaced them entirely or included additional references. We agree that we might have excluded important studies on the status of OA for individual disciplines. We now take into account disciplinary studies in the narrative review and in the Discussion section, thereby framing the results of the meta-synthesis. We now discuss the heterogeneity of included studies and how this might affect the consistency and comparability of study results (please see Methods, Results, Discussion). We now discuss the heterogeneity of included studies and how this might affect the comparability of study results (please see Methods, Results, Discussion). We have added a sub-section in which we state our definition of OA and its routes. In Table 2, we have added a column on the OA definition used by each study included in our review. Throughout the Results and the Discussion section, we note if included bibliometric studies differ from our definition of OA. In the Results section, we highlight how studies differed from one another in their definitions of OA and its sub-types. In the Discussion, we highlight the consequences thereof for the comparability of study results. In the narrative review, we now offer potential explanations for the popularity of Hybrid OA in specific disciplines. We have corrected the following typos: “66& for publication years…” - use %; replace “SEM” by “STM” for “Science, Technology and Medicine”; replace “PLoS” by “PLOS\" Due to sparse data on the early uptake on OA in the humanities, we have removed the conclusion regarding the comparison of OA levels for medical research areas and the humanities (p. 4 in version 1). We note this limitation in the narrative review. In Table 2, we have added a column on the OA definition used by each study included in our review. Throughout the Results and the Discussion section, we note if included bibliometric studies differ from our definition of OA. We added \"with DOIs\" in column two of^Piwowar et al (2018). We have cited the reference European Commission (2014) as Archambault et al. (2014). Table 3 (now Table 4): In a footnote, we added the information that for Piwowar et al. (2018), the figures for the Humanities and the Arts were not included as these disciplines are underrepresented in the WoS and in terms of DOI coverage. We have corrected the Hybrid OA overall figure for Piwowar et al. (2018). We have corrected the Hybrid OA overall figure for Piwowar et al. (2018), as well as the figures for Jamali and Navabi (2015). As for Martin-Martin et al. (2018) and Archambault et al. (2014), the sum of shares for individual OA routes does not match with the overall OA figure because both studies included Free Availability / Other OA in their estimation of OA levels – which this study does not. For both studies, we have noted this in footmarks and are discussing it in the Results and Discussion section. We have added two references to the statement “… as the OA model is unlikely to generate the level of income and profit that can be achieved with the subscription model”: Hagner (2008) and Johnson et al. (2017). For the statement “This also applies to academic and professional societies”, we have replaced the reference, now: Albert (2006). Table 4 (now Table 5): We have replaced “APC levels” with “publishing costs”. We also added more information on support aspects and copyright factors in Table 4. We now mention the year in which the NIH OA mandate was introduced. We now mention Wellcome Open Research. The remarks on OA and preprints in biology are now more differentiated. We agree that we should have elaborated on what “enjoy popularity” means in the context of OA journals in Chemistry. Shortening and restructuring of the paper however removed the entire statement from the text. We now provide two references for the stated national focus of engineering: Rostan et al. (2014) and Kyvik & Ingvild (2017)S Kyvik and R Ingvild. We have removed the bold face for the word \"Reflecting\". We have removed the redundancies in the introduction o “Open access in the social sciences”. and added more references for the order of OA routes. We have added a more recent reference for a statement about the present state of open access in the humanities: Rodriguez (2014). We have corrected the typo “Open Library of the Humanities\" We have removed the stament \"While long-term access to research outputs is questionable in these models…” We have rephrased the statement on growth of repositories in the social sciences to “Some attempts have been made to promote repositories in the social sciences” and provided two references for it: OpenDOAR database and Xia (2007). We have rephrased the statement “Because OA preprint repositories do not employ peer review, however, social scientists have been slow to adopt Green OA.” and now provide a reference for it. We have added references to the cited order of OA routes for the humanities. We have corrected the statement on MLA Commons. Shortening and restructuring the paper removed the following statement from the text: “post-publication peer review remains rare … with a few notable notable exceptions and experiments”. We have added an indicative reference for the statement that Plan S does not allow any embargoes. We have added references to the cited order of OA routes for law. We have corrected the statement that JIPITEC is based in the EU. We have corrected the following statements: “overall OA levels well above 50%”, “hybrid OA generally is of little variance”, “hybrid OA… with 1% or less of all scholarly outputs” We have added more detailed information on preprint uptake in biology to the section on OA in biology and added a respective reference: Kaiser (2017). Shortening and restructuring of the paper removed the following statement from the text: “Barriers to OA in chemistry and engineering can be identified as concerns about the quality of OA journals” Shortening and restructuring of the paper removed the following statement from the text: \"difficulties in assessing funding for APCs and BPCs”. Shortening and restructuring of the paper removed the following statement from the text: “we observed signs of cultural change particular in young scholars” Shortening and restructuring of the paper removed the following statement from the Discussion: “Most OA within the humanities is published as Hybrid OA…”. We however give a potential explanation for the popularity of Hybrid OA in the humanities in our narrative review. We added the reference Bulock, C. (2018) to the following statement: “This includes models such as OLH or other crowd funding initiatives, such as KU.” Shortening and restructuring of the paper removed the following statement from the text: \"… OA … is shaped by the scholars that use respective communication technologies.”"
}
]
}
] | 1
|
https://f1000research.com/articles/7-1925
|
https://f1000research.com/articles/9-81/v1
|
04 Feb 20
|
{
"type": "Software Tool Article",
"title": "The iCRF Generator: Generating interoperable electronic case report forms using online codebooks",
"authors": [
"Sander de Ridder",
"Jeroen A.M. Beliën"
],
"abstract": "Semantic interoperability of clinical data is essential to preserve its meaning and intent when the data is exchanged, re-used or integrated with other data. Achieving semantic operability requires the use of a communication standard, such as HL7, as well as (functional) information standards. Manually mapping clinical data to a medical thesaurus, such as SNOMED CT, is complicated and requires expert knowledge of both the dataset, including its context, and the thesaurus. As an alternative, the (re-)use of codebooks, data definitions which may already have been mapped to a thesaurus, can be a viable approach. We’ve developed the iCRF Generator, a Java program that can generate the core of an interoperable electronic case report form (iCRF) for several of the major electronic data capture systems (EDCs). To build their CRFs, users can select one or more items from established codebooks, available from an online system called ART-DECOR. By providing an easy to use method to create CRFs for multiple EDCs based on the same codebooks, interoperability can be more easily attained.",
"keywords": [
"Interoperability",
"eCRF",
"iCRF",
"Codebook",
"FAIR",
"Software",
"EDC",
"Clinical data"
],
"content": "Introduction\n\nClinical data is essential for health research. Traditionally, such data was captured using paper case report forms (CRFs) and entered into a database manually. Nowadays, the data is often captured directly with electronic CRFs (eCRFs) in an electronic data capture (EDC) system. This has improved the quality of the captured data as well as decreased costs for data collection (e.g. 1,2).\n\nTo allow the captured data to be used beyond its original purpose requires the data to be FAIR (Findable, Accessible, Interoperable and Reusable)3. By making data semantically interoperable, it can be exchanged between systems whilst preserving the meaning of the data4. Furthermore, it allows multiple data sources to be combined and understood by computers, thereby e.g. facilitating clinical decision support systems5. Hence, when setting up a new data collection protocol, the eCRF should be designed with interoperability in mind. Achieving semantic interoperability requires the use of a communication standard, such as HL7, as well as (functional) information standards4, such as the NCI thesaurus or SNOMED CT. However, mapping study-specific terminology to a thesaurus requires expert knowledge of the thesaurus, the data and its context. Therefore, reusing existing codebooks from studies and well-known datasets or CRF templates, such as available from CDISC’s CDASH, the Meta Data Models website and the University of Wisconsin-Madison, can be a viable alternative. Reusing these elements at the very minimum facilitates interoperability with other datasets using these definitions. Furthermore, in many cases well-known codebooks have already been mapped to a thesaurus. For example, in the Basic Health Data Set, which is the standard that will be used by hospitals to exchange healthcare data in the Netherlands (available here, Dutch only), many of the items have been mapped to SNOMED CT.\n\nIn this paper, we introduce the iCRF Generator, a program that allows users to easily generate interoperable electronic case report forms (iCRFs) based on online codebooks, thereby improving the interoperability of clinical data collected in and between EDCs. Whereas normally CRF generation is an integrated part of the EDC (e.g. Castor EDC, REDCap), our program can generate the core of a CRF for multiple EDCs. At this time, three systems are supported: Castor, OpenClinica 3 and REDCap. The program allows a user to select one or more codebooks available from an online system called ART-DECOR which allows, amongst others, the storage of dataset definitions and select items of interest, including their codelists. The program currently supports six codebooks, which are further described in the Methods section.\n\n\nMethods\n\nThe iCRF Generator was written in Java 8 and later migrated to Java 12 for JavaFX compatibility. Dependencies are managed using Maven and include: JavaFX and ControlsFX for the UI, Apache POI for Excel file management and Log4j for logging. A ZIP file of the iCRF Generator distribution is available for both Mac and Windows. It includes a Java Runtime Environment to ensure independence of the locally installed Java version and ensures the program works out of the box. Source and distribution files are available on GitHub: https://github.com/aderidder/iCRFGenerator/.\n\nThe iCRF Generator is designed to use codebooks defined in ART-DECOR. ART-DECOR is an open-source tool suite that supports the creation and maintenance of HL7 templates and allows the storage of dataset definitions. Nictiz, the centre of expertise for eHealth and the Dutch SNOMED-CT release centre, facilitates ART-DECOR to create health information standards that are publicly accessible. The iCRF Generator currently offers access to six of these codebooks, which were chosen because of their national relevance (codebooks 1, 2 and 3) and our involvement (4, 5 and 6):\n\n1. The Clinical Building Blocks (Zorginformatiebouwstenen): information models of minimal clinical concepts. They are used as the basis for the Basic Health Data Set.\n\n2. The Basic Health Data Set (Basisgegevensset Zorg): codebook used for the standardised exchange of patient data between e.g. healthcare providers. Implementation of this set is prioritised in healthcare systems like electronic health records. The Basic Health Data Set is aligned with the European Patient Summary.\n\n3. The National Institute for Public Health and the Environment’s national screening codebook of bowel cancer and cervical cancer (RIVM bevolkingsonderzoeken).\n\n4. Cancer Core Europe: a European cancer research alliance which aims at bringing together the expertise and critical mass necessary to make translational research available in the clinic.\n\n5. The PALGA Colon biopsy protocol: PALGA is the nationwide network and registry of histo- and cytopathology in the Netherlands.\n\n6. The PALGA Colorectum carcinoma protocol.\n\nSome of the codebooks are available in English, as well as Dutch. Other codebooks may be made available in the iCRF Generator in the future when they become available in ART-DECOR provided they are of sufficient quality and are complementary.\n\nA standard PC or Mac should be able to run the program without any issues. The program was tested on a Windows 7 PC, a Windows 10 PC and on a virtual machine with OS X El Capitan. To give an indication of the program’s memory usage: selecting a single codebook, the program uses around 230 megabytes of memory; increasing this number to eight codebooks increased the memory usage to 420 megabytes. An internet connection is required, as the program retrieves metadata as well as codebooks from ART-DECOR via the REST API.\n\n\nUse cases\n\nA typical use case for the iCRF Generator is in the design phase of a study or registry. When a decision has been made on what clinical data is going to be collected, the data manager has to design and build the CRFs for data collection. Instead of doing this manually, the iCRF Generator can be used to select items from the available codebooks and generate the basis of the case report forms. Figure 1 illustrates the iCRF Generator’s complete workflow. In a typical use case, the user first selects an EDC from the dropdown (Figure 2). The user then clicks the “Run” button, after which the wizard interface is started. The first wizard page asks the user to select one or more codebooks (Figure 3). When a user has selected a codebook and presses the “Next” button, a REST-call is made to retrieve an XML which contains which versions and languages are available for the codebook. The second page shows this information to the user, allowing selection of the versions and languages of interest (Figure 4). When the user proceeds to the next page, the selected codebooks are retrieved via one or more REST-calls. These XML files are parsed and the information it contains about the items and their possible values is shown on the third page (Figure 5). This EDC-specific page allows users to select and customise the items that have to be included in the CRF. The final page of the wizard shows a short summary of the number of selected items. Upon completion of the wizard, the program generates the CRF in the format required by the selected EDC and the file is saved to disk. This file can then be imported into the EDC-system or opened in an editor of choice.\n\nDotted lines are calls made by the program to ART-DECOR’s REST services.\n\n\nDiscussion\n\nTo allow for the use of data beyond its original purpose, it is essential that the data is FAIR (Findable, Accessible, Interoperable and Reusable)3. To preserve meaning and intent of clinical data when it is exchanged, requires the data to be semantically interoperable, which requires the use of content standards. Manually mapping data definitions to a medical thesaurus such as SNOMED CT is complicated and time consuming. Instead, reusing existing published definitions (codebooks) can be a viable alternative, as interoperability with other datasets using these codebooks is usually easily achieved. Furthermore, the codebooks may already have been mapped to a thesaurus.\n\nIn this paper we introduced the iCRF Generator, a program which can generate electronic case report forms for three major electronic data capture systems: Castor, REDCap and OpenClinica 3. The program allows a user to select items and codelists from several highly relevant codebooks available from the online system ART-DECOR. By providing an easy to use program to generate CRFs using these codebooks, data will be collected using the same definitions, which enhances the interoperability and FAIRness of the data.\n\nOne important usability aspect of software is the software’s performance. The codebooks available in the iCRF Generator are parsed from XML files generated by ART-DECOR. It takes ART-DECOR around 30 seconds to generate the XML file for the National Institute for Public Health and the Environment screening codebook. Furthermore, in some cases XML files can reference other XML files, which then have to be downloaded and parsed as well. If a user has to wait every time a codebook is selected, user acceptance will quickly erode. Hence, we introduced local caching of downloaded codebooks, which makes usage nearly instantaneous once the codebook is locally available. Furthermore, we intend to make a ZIP file of the cache available for download. Note that downloading a codebook XML from ART-DECOR only takes place if it is accessed for the first time or when a new version of a codebook becomes available and is selected by the user.\n\nThe iCRF Generator can easily be expanded to include additional EDCs, such as OpenClinica 4, Research Manager and Alea if there is demand and the import formats are available. If desirable, additional internationally established formats, such as CDISC ODM, may also be included in the future.\n\nAt this point, the iCRF Generator gives access to six codebooks, some of which support multiple languages and multiple versions. When more codebooks are published in ART-DECOR, we intend to make these available in the program, provided they are of sufficient quality and are complementary to the codebooks already available. Criteria to decide whether a codebook will be made available will be established in the near future. We believe that providing too many overlapping codebooks will be counterproductive for interoperability as items may well map to different thesauri. Furthermore, the user may be overwhelmed by the available information.\n\nWhen an item is selected in the item tree, a user can customise the item. As each EDC has different requirements for its CRFs, the customisation options we provide vary per EDC. As an example, OpenClinica 3 has a “Field Type” (e.g. “Radio”, “Single-Select”) and a “Data Type” (e.g. “ST”, “INT”), whereas in Castor the data type does not exist as a separate entity.\n\nThe customisation options we currently provide are limited. The iCRF Generator’s purpose is to facilitate generation of interoperable CRFs. Hence, if everything could be customised, for example replacing the codes in codelists with custom codes, it would undermine the purpose of the program. Furthermore, the iCRF Generator is work-in-progress and some further item customisation may be added in the future.\n\nA tool somewhat similar to our own is ODMedit6. ODMedit provides a web-based interface to allow users to create a CRF based on elements stored in the Meta Data Repository. When a user has finished creating the CRF, it can either be downloaded in ODM format or uploaded to the Medical Data Models-portal. From there it can be downloaded in multiple formats.\n\nODMedit differs from our software in several ways. Whereas ODMedit immediately provides access to all items in its repository, we keep our items grouped by codebook. Furthermore, with ODMedit users can immediately add new and edit existing items, and new items are automatically made available in the repository. In our tool we are providing access to only handpicked codebooks, from which the user can select items and customisation of these items is kept to a bare minimum. By allowing users to select items from well-known and supported codebooks only, we believe it should be easier to find the correct item - e.g. if you need pathology definitions, use items from the pathology codebooks. However, we may have to add a search function at some point to make it easier to find items within a codebook. Another difference is that we decided to explicitly ask for which EDC tool the user wishes to create the CRF to allow for EDC specific options. On the other hand, ODMedit does support some features which we do not yet support, such as a repeating group. We may add this at a future time.\n\nAn OpenClinica 3 specific CRF generator is also available. This tool converts a csv file to Excel and provides a user with an interface to edit the CRF. However, the tool does not facilitate interoperability.\n\nMultiple initiatives exist that aim at providing templates to improve interoperability. We list several such initiatives below. The National Institute of Health offers Common Data Elements, data elements that are common to multiple data sets across different studies. CDASH, provided by CDISC, gives guidance for developing CRFs used in clinical trials7. The OpenClinica Building Blocks developed by TraIT provide OpenClinica users with templates to which they can add study-specific items and remove items that are not necessary for their study. The Australian Government launched a platform for digital health. They provide an extensive library of documents, tools and much more for implementers and developers. The Global Alliance for Genomics & Health (GA4GH) has several workstreams, amongst which one for Clinical & Phenotypic Data Capture, that “Supports the clinical adoption of genomics through establishing standard ontologies and information models to describe the clinical phenotype for use in genomic medicine and research, including the capture and exchange of information between electronic clinical systems and research.”\n\n\nSoftware availability\n\nSource code available here: https://github.com/aderidder/iCRFGenerator\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.35635008\n\nLicense: GNU GPL v3 license.",
"appendix": "Acknowledgements\n\nWe thank Jan-Willem Boiten (Lygature), Gerben Rienk Visser (Trial Data Solutions) and Maarten Ligtvoet (Nictiz) for reviewing this paper and providing invaluable suggestions. We also thank Wessel Sloof (UMCG) for testing the generated REDCap exports.\n\n\nReferences\n\nSahoo U, Bhatt A: Electronic data capture (EDC)--a new mantra for clinical trials. Qual Assur. 2003; 10(3–4): 117–21. PubMed Abstract | Publisher Full Text\n\nThriemer K, Ley B, Ame SM, et al.: Replacing paper data collection forms with electronic data entry in the field: findings from a study of community-acquired bloodstream infections in Pemba, Zanzibar. BMC Res Notes. 2012; 5(1): 113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshrafi N, Kuilboer JP, Stull T: Semantic Interoperability in Healthcare: Challenges and Roadblocks. In STPIS@ CAiSE. 2018; 119–122. Reference Source\n\nAhmadian L, van Engen-Verheul M, Bakhshi-Raiez F, et al.: The role of standardized data and terminological systems in computerized clinical decision support systems: literature review and survey. Int J Med Inform. 2011; 80(2): 81–93. PubMed Abstract | Publisher Full Text\n\nDugas M, Meidt A, Neuhaus P, et al.: ODMedit: uniform semantic annotation for data integration in medicine based on a public metadata repository. BMC Med Res Methodol. 2016; 16(1): 65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaddale JR: Clinical Data Acquisition Standards Harmonization importance and benefits in clinical data management. Perspect Clin Res. 2015; 6(4): 179–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Ridder S: aderidder/iCRFGenerator: First release (Version V1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3563500"
}
|
[
{
"id": "59542",
"date": "07 Feb 2020",
"name": "Rianne Fijten",
"expertise": [
"Reviewer Expertise Our area of research focuses on making medical data FAIR. For example",
"we employ the Personal Health Train to share information across medical centers in a privacy-preserving manner."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall The authors' idea of creating an iCRF generator could alleviate pressure from data managers by making CRFs interoperable.\nIntroduction The authors have presented all relevant concepts needed to understand the rest of the article.\nMethods\nCodebooks: Only 2 of 6 currently supported codebooks are generic enough to be used by users from a wide range of fields. Because the other four are all related to cancer, this could exclude a large group of users.\n\nCreating the CRFs: One of the arguments made in the article is that automating CRF will help people make CRFs more easily. However it is unclear whether using the CRF generator would actually save time. In fact, the figures imply that using the iCRF generator requires a lot of manual work as well.\nFigure 1:\nIn this figure, it is at first not clear where to start. We recommend using numbering to clarify the sequential steps.\n\nIt is unclear from the figure and text how the workflow is structured. In fact, the figure seems to imply that there are three different workflows depending on the EDC that is selected by the user. This could be a potential hazard in terms of sustainability as adding an EDC would be a lot of work. A generic workflow with EDC-specific export functionalities would make the program much more future-proof.\n\nWhat does the summary show?\nFigure 3 To help users, we suggest to implement a way of providing information about the codebooks and their content. For example, a link to more information about each codebook or a dropdown view that shows the items in each codebook. This is not there currently and would require users to start searching for it themselves if they're not familiar (enough) with the content of the codebooks.\nUse cases We would like to see a clearer example that also shows what each step in figure 1 looks like.\nDiscussion\nThe discussion mentions that codebooks might be added when of sufficient quality. How do you define sufficient quality and who decides that?\n\nThe authors mention that ODMedit's flexibility is a weakness compared to the iCRF generator. We believe that this could in fact be a strength if introduced in the iCRF generator. In fact, allowing users to contribute to the codebooks and item lists would widen the user base. To avoid the creation of faulty or multiple items denoting the same term, the authors could consider allowing users to contribute, but establish a curated group of codebooks and elements. This makes the software tool more flexible for its users.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "5309",
"date": "23 Mar 2020",
"name": "Sander de Ridder",
"role": "Author Response",
"response": "Thank you reviewing of our paper. Please find below the changes we made to the new version to address your concerns/comments. Methods 1. Codebooks: Only 2 of 6 currently supported codebooks are generic enough to be used by users from a wide range of fields. Because the other four are all related to cancer, this could exclude a large group of users. Indeed, currently the number of codebooks available is limited. We are hoping that by introducing the iCRF Generator to the public, this will stimulate them to publish codebooks in ART-DECOR, which we could then potentially make available in the iCRF Generator. Furthermore, Nictiz is also actively involved; they are currently looking into publishing e.g. established questionnaires. Given the comments here and later on, we rewrote the “Additional codebooks” paragraph in the discussion to: At this point, the iCRF Generator gives access to six nationally established codebooks, some of which support multiple languages and multiple versions. To improve the user-base for the iCRF Generator, the number and variety of codebooks available must increase. While nationwide standards, such as the Basic Health Dataset, can be readily made available, some form of governance may have to be put in place to establish other types of curated codebooks. This could stimulate the community to help build high-quality codebooks, mapped to medical thesauri, while preventing too much codebook redundancy and conflicting items. Furthermore, internationally established codebooks, such as CDISC’s CDASH can also be made available. 2. Creating the CRFs: One of the arguments made in the article is that automating CRF will help people make CRFs more easily. However it is unclear whether using the CRF generator would actually save time. In fact, the figures imply that using the iCRF generator requires a lot of manual work as well. It makes creating interoperable CRFs easier, not necessarily CRFs. Instead of having to e.g. define Gender and the appropriate codes and manually annotating with the appropriate thesaurus codes, which really takes a lot of time, instead you take the gender from the codebook and reuse that definition. However, you still need to define whether you would like have Gender as a radio button or a dropdown – that work does not change. We checked the manuscript and updated the Use cases section to stress this aspect: \"When a decision has been made on what clinical data is going to be collected, the data manager has to design and build the CRFs for data collection. Instead of doing this manually, the iCRF Generator can be used to select items from the available codebooks and generate the basis of the case report forms.\" Rewritten to \"When a decision has been made on what clinical data is going to be collected, the data manager has to design and build the CRFs for data collection. Instead of manually designing the items and mapping them to a medical thesaurus, which takes a lot of time, the iCRF Generator can be used to select items which have already been mapped from the available codebooks and generate the basis of the case report forms.\" 3. Figure 1 - In this figure, it is at first not clear where to start. We recommend using numbering to clarify the sequential steps. New version included in paper in which we numbered the steps 4. It is unclear from the figure and text how the workflow is structured. In fact, the figure seems to imply that there are three different workflows depending on the EDC that is selected by the user. This could be a potential hazard in terms of sustainability as adding an EDC would be a lot of work. A generic workflow with EDC-specific export functionalities would make the program much more future-proof. Indeed, it is a valid point that each EDC has its own customisation page, which thereby comes at the cost of maintainability / sustainability. However, this was done to facilitate the data managers. As an example, Castor's import file is an XML file, which you don't want to edit manually. If we remove all customisation options from de iCRF Generator, this implies all fields will have to be generated with default settings, or the XML will be invalid, e.g. every selection item will be a \"required dropdown\". This means that a datamanager would import these fields and would then have to manually edit all imported fields in Castor. We believe this would not make for a good user experience. We added the following to the \"EDC-specific item customisation within iCRF Generator\" section to clarify our reasoning: By adding this EDC-specific customisation, the iCRF Generator’s code is more difficult to maintain. However, by allowing the data manager to customise essential fields, the iCRF Generator can provide a ready-to-use CRF. This enhances the user experience, making it a worthwhile investment. 5. What does the summary show? In the text we mention the following about the summary: “The final page of the wizard shows a short summary of the number of selected items.” We’ve added a new Figure showing a screenshot of the summary. 6. Figure 3 - To help users, we suggest to implement a way of providing information about the codebooks and their content. For example, a link to more information about each codebook or a dropdown view that shows the items in each codebook. This is not there currently and would require users to start searching for it themselves if they're not familiar (enough) with the content of the codebooks. Great idea. We could change the page where you can select the codebooks and make the codebooks themselves e.g. clickable, linking to their ART-DECOR page. We'll add this to the roadmap for a future release. Use cases 7. We would like to see a clearer example that also shows what each step in figure 1 looks like. To clarify the relationship between Figure 1 and the example figures (2-6) we updated the text to show how each step in Figure 1 is related to the example figures: Figure 1 illustrates the iCRF Generator’s complete workflow and figure 2-6 show actual examples of the workflow. In a typical use case, the user first selects an EDC from the dropdown ( Figure 1, step 1; Figure 2). The user then clicks the “Run” button, after which the wizard interface is started. The first wizard page asks the user to select one or more codebooks (Figure 1, step 2; Figure 3). <etc> Discussion 8. The discussion mentions that codebooks might be added when of sufficient quality. How do you define sufficient quality and who decides that? Our initial aim was to provide a tool which can facilitate the reuse of the available codebooks. How to provide the users with high-quality codebooks is open for discussion and we welcome any input on the matter. We did rewrite this section though - see point 1. 9. The authors mention that ODMedit's flexibility is a weakness compared to the iCRF generator. We believe that this could in fact be a strength if introduced in the iCRF generator. In fact, allowing users to contribute to the codebooks and item lists would widen the user base. To avoid the creation of faulty or multiple items denoting the same term, the authors could consider allowing users to contribute, but establish a curated group of codebooks and elements. This makes the software tool more flexible for its users. Any codebook can be added to ART-DECOR, as Nictiz welcomes any additions. To facilitate interoperability, we ourselves foremost welcome any (inter)nationally endorsed standard, such as the Basic Health Dataset. We agree that establishing a curated group of codebooks which are properly mapped to medical thesauri would be great, although this does require a proper decision-making structure (governance) as well as the appropriate funding. We rewrote this section - see point 1."
}
]
},
{
"id": "59545",
"date": "14 Feb 2020",
"name": "Martin Dugas",
"expertise": [
"Reviewer Expertise Medical Informatics",
"Medical Data Models",
"semantic annotations",
"interoperability"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe iCRF Generator: Generating interoperable electronic case report forms using online codebooks\nThis manuscript is about iCRF generator, a software tool to generate interoperable CRFs. To foster re-use of data and implement FAIR principles are important topics. Source code of iCRF-generator is available, which is a plus.\nMajor comments page 2, Methods, Supported Codebooks: Quantitative Information on 6 codebooks is missing: how many item definitions per codebook are available? how many codelist(items)?\npage 2, Introduction: iCRF generates CRFs in Castor, OpenClinica3 and REDCap-Format. Why not CDISC ODM, which is endorsed by FDA? The Portal of Medical Data Models provides CRFs in 18 formats\npage 3, Discussion \"Manually mapping data definitions to a medical thesaurus such as SNOMED CT is complicated and time consuming\" => Yes, this is absolutely correct. But it should be mentioned, that this mapping was already done for large sets of data items. For example, the portal of medical data models provides ~520.000 data items with manual (physician-based) terminology mappings. The portal of medical data models is not just a website, it is a registered European information infrastructure.\npage 6, Additional EDCs: CDISC ODM is not just an internationally established format, it is required by regulatory authorities (FDA). Define XML (FDA) is based on CDISC ODM !\npage 6, EDC-specific item customisation: processing of individual data items is a very work-intensive process, given the high number of data items in clinical studies (~ 500 - 2000). Re-Use of itemgroups (sets of data items) would be useful (this feature is available in ODMedit, which is covered in the discussion)\npage 6, Discussion, similar work: It should be mentioned, that ODMedit provides semantic coding (especially UMLS codes) for data items and codelists. Is semantic coding available in iCRF?\nMinor comments page 2, Introduction, 2nd paragraph: HL7 is not a communication standard, but a standards developing organisation\npage 2, Introduction, 2nd paragraph: the \"Meta Data Models website\" correct term: Portal of Medical Data Models\npage 2, Introduction, 2nd paragraph: Link to Basic Health Data Set (available contents!) should be updated\nFigure 2 & Figure 4: should be shrinked, a lot of blank space\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": [
{
"c_id": "5310",
"date": "23 Mar 2020",
"name": "Sander de Ridder",
"role": "Author Response",
"response": "Thank you reviewing of our paper. Please find below the changes we made to the new version to address your concerns/comments. Major comments 1. page 2, Methods, Supported Codebooks: Quantitative Information on 6 codebooks is missing: how many item definitions per codebook are available? how many codelist(items)? We added this information to the “supported codebooks” section. 2. page 2, Introduction: iCRF generates CRFs in Castor, OpenClinica3 and REDCap-Format. Why not CDISC ODM, which is endorsed by FDA? The Portal of Medical Data Models provides CRFs in 18 formats One of the issues is that Castor doesn't support it yet. OpenClinica 3 doesn’t support direct import of ODM CRFs either. Hence, supporting ODM wasn’t a priority for us in the current release, but given your feedback, we added support for CDISC ODM to the roadmap. Accordingly, we updated: \"If desirable, additional internationally established formats, such as CDISC ODM, may also be included in the future.\" in the “Additional EDCs” section to: \"Support for CDISC ODM is on the roadmap. If desirable, additional internationally established formats may also be included in the future.\" 3. page 3, Discussion \"Manually mapping data definitions to a medical thesaurus such as SNOMED CT is complicated and time consuming\" => Yes, this is absolutely correct. But it should be mentioned, that this mapping was already done for large sets of data items. For example, the portal of medical data models provides ~520.000 data items with manual (physician-based) terminology mappings. The portal of medical data models is not just a website, it is a registered European information infrastructure. We updated the manuscript to stress that many items have already been mapped. 4. page 6, Additional EDCs: CDISC ODM is not just an internationally established format, it is required by regulatory authorities (FDA). Define XML (FDA) is based on CDISC ODM ! As mentioned in an earlier comment, CDISC ODM can be added, but it wasn't a priority for us. However, we added it to the roadmap. 5. page 6, EDC-specific item customisation: processing of individual data items is a very work-intensive process, given the high number of data items in clinical studies (~ 500 - 2000). Re-Use of itemgroups (sets of data items) would be useful (this feature is available in ODMedit, which is covered in the discussion) Thank you for the suggestion. We’d have to look into it more closely, but we added it as a possible new feature to the roadmap. 6. page 6, Discussion, similar work: It should be mentioned, that ODMedit provides semantic coding (especially UMLS codes) for data items and codelists. Is semantic coding available in iCRF? The iCRF Generator is independent of the coding used. Hence, if a codebook there uses UMLS, the iCRF Generator will show UMLS codes. We added the following to the introduction: Hence, if a codebook is mapped to a medical thesaurus, the iCRF Generator allows the user to use these mappings, preventing the labour-intensive manual mapping. Minor comments 7. page 2, Introduction, 2nd paragraph - HL7 is not a communication standard, but a standards developing organisation Health Level Seven International (HL7) is the standards developing organization. However, Health Level Seven (HL7) also refers to the standards. See e.g. their glossary here: https://www.hl7.org/documentcenter/public/calendarofevents/FirstTime/Glossary%20of%20terms.pdf Health Level Seven (HL7) is an application protocol for electronic data exchange in health care environments. The HL7 protocol is a collection of standard formats which specify the implementation of interfaces between computer applications from different vendors. This communication protocol allows healthcare institutions to exchange key sets of data amount different application systems. Flexibility is built into the protocol to allow compatibility for specialized data sets that have facility specific needs. To avoid confusion, we updated the URL in the paper to directly link to the standards (https://www.hl7.org/implement/standards/ ). 8. page 2, Introduction, 2nd paragraph: the \"Meta Data Models website\" correct term: Portal of Medical Data Models We apologise for the error. Fixed. 9. page 2, Introduction, 2nd paragraph: Link to Basic Health Data Set (available contents!) should be updated Thank for the suggestion. We added the URL. 10. Figure 2 & Figure 4: should be shrinked, a lot of blank space We removed blank space from the figures."
}
]
}
] | 1
|
https://f1000research.com/articles/9-81
|
https://f1000research.com/articles/9-200/v1
|
23 Mar 20
|
{
"type": "Research Article",
"title": "Accuracy of short tandem repeats genotyping tools in whole exome sequencing data",
"authors": [
"Andreas Halman",
"Alicia Oshlack",
"Andreas Halman"
],
"abstract": "Background: Short tandem repeats are an important source of genetic variation. They are highly mutable and repeat expansions are associated dozens of human disorders, such as Huntington's disease and spinocerebellar ataxias. Technical advantages in sequencing technology have made it possible to analyse these repeats at large scale; however, accurate genotyping is still a challenging task. We compared four different short tandem repeats genotyping tools on whole exome sequencing data to determine their genotyping performance and limits, which will aid other researchers in choosing a suitable tool and parameters for analysis. Methods: The analysis was performed on the Simons Simplex Collection dataset, where we used a novel method of evaluation with accuracy determined by the rate of homozygous calls on the X chromosome of male samples. In total we analysed 433 samples and around a million genotypes for evaluating tools on whole exome sequencing data. Results: We determined a relatively good performance of all tools when genotyping repeats of 3-6 bp in length, which could be improved with coverage and quality score filtering. However, genotyping homopolymers was challenging for all tools and a high error rate was present across different thresholds of coverage and quality scores. Interestingly, dinucleotide repeats displayed a high error rate as well, which was found to be mainly caused by the AC/TG repeats. Overall, LobSTR was able to make the most calls and was also the fastest tool, while RepeatSeq and HipSTR exhibited the lowest heterozygous error rate at low coverage. Conclusions: All tools have different strengths and weaknesses and the choice may depend on the application. In this analysis we demonstrated the effect of using different filtering parameters and offered recommendations based on the trade-off between the best accuracy of genotyping and the highest number of calls.",
"keywords": [
"short tandem repeats",
"microsatellites",
"gangstr",
"lobstr",
"hipstr",
"repeatseq"
],
"content": "Introduction\n\nSTRs, also known as microsatellites, consist of repeated units of 1 to 6 base pairs (bp) in length and cover about 3% of the human genome (Gymrek, 2017). STRs are highly mutable and often vary in their number of repeat units across the population. They can be found in various regions of the genome, including in or near protein coding regions and introns (Hannan, 2018). Expanded variants contribute to several dozen human disorders, including Huntington's disease, fragile X syndrome, spinocerebellar ataxias and other diseases. In addition, variation in STR length has been shown to associate with quantitative traits such as gene expression (Gymrek, 2017). The standard method to genotype the length of STRs is to perform polymerase chain reaction (PCR) amplification on the region of interest and gel electrophoresis (Tang & Nzabarushimana, 2017). Sanger sequencing has high accuracy, but low throughput, limiting analysis to a few genes at a time (Caspar et al., 2018).\n\nRecent technology advances in high-throughput sequencing (HTS) have revolutionised the genomics field and brought us the opportunity to detect sequence variants at a scale that was impossible before (Caspar et al., 2018). HTS of the whole genomes provides the potential to profile over a million STRs in the human genome. Recent advances in bioinformatics have brought us tools to analyse STRs from sequencing data (HTS), but genotyping still remains challenging for many reasons, including issues with extreme GC content, short read lengths that do not span over the entire repeat, and issues with alignment due to variation in STRs appearing as large insertions or deletions relative to the reference. In addition, using PCR amplification during library preparation will often cause stutter noise and produce artificial variability in the sequence (Caspar et al., 2018; Gymrek, 2017). Stutter noise is a result of in vitro slippage of DNA polymerase during PCR cycles that leads to erroneous reads of incorrect repeat length (Willems et al., 2014), which contributes to challenges in genotyping.\n\nIllumina has developed a method for amplification-free (PCR−) library preparation (Kozarewa et al., 2009), which theoretically eliminates the STR stutter error during PCR amplification in sample preparation (PCR+) and therefore improves the accuracy of STR genotyping. The developers of STR-FM evaluated the new protocol by running their tool in both PCR− and PCR+ samples and found that the PCR− protocol compared to PCR+ has up to nine-fold fewer errors (Fungtammasan et al., 2015). However, huge amounts of sequencing data have already been generated by using the PCR+ protocol, where some data will not be resequenced due to time and/or cost (Fungtammasan et al., 2015). In addition, despite the advantages of whole genome sequencing (WGS), whole exome sequencing (WES) is still widely used in human genetics due to its lower cost and higher coverage and WES is a PCR+ process (Björn et al., 2018). Therefore, tools that can accurately genotype STRs not only from PCR− but also from PCR+ data are essential.\n\nWhile there are a number of computational tools that have been developed to genotype STR alleles in HTS data, there have been few independent comparisons of their performance. Evaluation of methods for genotyping STRs is difficult. The gold standard measurement of STRs is by capillary electrophoresis (Willems et al., 2014), but these methods have low throughput. Further evaluations have used Mendelian inheritance as a measure of accuracy (Gymrek et al., 2012; Highnam et al., 2013; Mousavi et al., 2019). Other studies have used simulated data for the evaluation of genotyping accuracy (Fungtammasan et al., 2015; Highnam et al., 2013). While simulation can generate many loci with known alleles, it is difficult to simulate the true complexity of real data.\n\nHere we propose to compare and evaluate STR genotyping methods on exome data using a different but complementary approach. We used the natural hemizygous state of the X chromosomes in males to look for incorrect calls revealed by a heterozygous call. With repeats on the X chromosome in males there is only one allele, so we expect all calls to be homozygous. While this approach does not evaluate the accuracy of the allele length, it has advantages in that (a) the data sets are large so we can test thousands of calls, and (b) the data comes from real patients with all the noise and biases found in real data.\n\nIn our study, we compared LobSTR (Gymrek et al., 2012), RepeatSeq (Highnam et al., 2013), HipSTR (Willems et al., 2017) and a recently published tool GangSTR (Mousavi et al., 2019). In addition, we included a common variant calling tool GATK HaplotypeCaller (McKenna et al., 2010) as a comparison of genotyping accuracy.\n\nThere are a number of tools that have been developed for STR analysis and which were excluded from this analysis. For example, popSTR (Kristmundsdóttir et al., 2017) is a population based STR genotyper and is optimised for whole genome sequencing (WGS) data. STRviper is another method for genotyping STRs that is able to pick up repeats longer than the read length; however, it has no built-in stutter model and it is not suitable for diploid dataset as it assumes only one allele (Cao et al., 2014). Galaxy environment has an STR analysis tool called STR-FM which we were unable to run (Fungtammasan et al., 2015). Dante (Budiš et al., 2019) and STRScan (Tang & Nzabarushimana, 2017) are designed for targeted searches and require a user-defined list of STR loci.\n\nTools such as Expansion Hunter (Dolzhenko et al., 2017; Dolzhenko et al., 2019), TREDPARSE (Tang et al., 2017), STRetch (Dashnow et al., 2018) and exSTRa (Tankard et al., 2018) were excluded from our analysis as well because they are classified as tools specifically looking for expansions that might be disease causing and are often longer than the physical read length or expansion relative to a control set.\n\nOur analysis focuses on comparing the performance of STR genotyping tools on the X chromosome of more than 400 males. Using this data set, we investigate the overall ability of tools to call genotypes, the accuracy as a function of coverage and repeat unit and also investigate quality scores of the tools. We find most tools are able to call a majority of homozygous alleles and different tools have different advantages in terms of repeat unit and coverage.\n\nFirst, we will give a short overview of STR genotyping tools included in our analysis and their reported accuracy. The tools evaluated in our analysis are summarised in Table 1. All of the tools require a set of defined STR loci. Tandem Repeats Finder (TRF) is a tool that can be used to detect STRs that have two or more copies of the same repeat unit in a row in the reference genome (Benson, 1999). In addition, it can detect repeats for which the repeat unit size is up to several hundred of bp long. Running TRF generates a report that includes all the loci detected in the genome, with genomic start and end location of the STR, repeat unit and its size, number of copies aligned with the consensus pattern and other relevant information. For this study, we limited the loci defined by TRF to repeat units up to 6 bp (see Methods).\n\nLobSTR. LobSTR was one of the first successful STR genotyping tools for HTS data. It initially used its own inbuilt aligner but can also use data aligned with BWA-MEM (Li, 2013). LobSTR identifies reads that completely contain the STR and which also have flanking sequence with no repetitive sequence when aligned to a reference genome. As mentioned, PCR amplification during library preparation can create stutter noise at an STR locus, and LobSTR tackles this issue with an included stutter model that aims to detect and account for noise to improve genotyping accuracy. The stutter noise model used can be custom generated from the data or the standardised one supplied by the tool developers. As a result, LobSTR determines and reports the maximum likelihood estimates of the genotype in each locus (Gymrek et al., 2012).\n\nLobSTR was validated using concordance of biological replicates (blood and saliva samples) from the same subject to measure the precision of the tool. At 21x coverage, the discordance rate for genotype was 3% and for allelotype was 2%. While for lower 5x coverage, the discordance rate for genotype was 11% and for allelotype was 5%. STR length differences were analysed in discordant calls that were heterozygous in both blood and saliva samples and found that at coverage 5x or higher, 90% of the errors were one repeat unit difference and 99% of errors were in 2 bp repeat unit size (Gymrek et al., 2012). However, it is important to note that LobSTR validated 2–6 bp repeat unit size STRs and did not validate homopolymers (Gymrek et al., 2012), which are common in the genome and a known source of genetic variation (Highnam et al., 2013).\n\nRepeatSeq. RepeatSeq (Highnam et al., 2013) uses data aligned by an external tool, such as BWA or Bowtie. It uses Bayesian model selection to determine the most probable genotype and requires all reads to fully contain STRs and at least two reads at a locus to make a call. The RepeatSeq noise model is based on genomes derived from over 100 inbred isolates of fly.\n\nRepeatSeq's accuracy was evaluated by analysing a trio WGS data to test consistency with Mendelian inheritance. The authors reported that on minimum coverage of two, 92.1% of repeat calls were consistent with the Mendelian inheritance, while with a minimum coverage of nine it was 95.3% and on minimum coverage of 17 it was 98.0% (Highnam et al., 2013).\n\nGangSTR. One of the major drawbacks of the first series of STR profiling methods was that they were limited to genotyping repeats within the read length in HTS data. GangSTR (Mousavi et al., 2019) is a more recent method that incorporates additional information besides repeat-enclosing reads to estimate the length of repeats. This includes available information such as fragment length, coverage and information about partially enclosing reads where only one end contains flanking sequence. More specifically, reads are divided into four classes: 1) enclosing read pairs that have at least one read that includes the whole STR and a flanking region in both ends; 2) spanning read pairs that have a mate pair where one read is aligned to one side of the STR and the second read of the pair on the other side; 3) flanking read pairs that include a read which partially extends into the STR region; and 4) fully repetitive read pairs that have one or two reads which are entirely made of STR (Mousavi et al., 2019). These four classes of reads are used to not only genotype repeats less than the read length but can also be used to genotype longer alleles such as repeat expansions.\n\nThe GangSTR method was evaluated by first simulating paired-end 150 bp reads (40x coverage) for 14 repeat expansions involved in STR disorders. Tool accuracy was measured by comparing true and observed alleles and also compared to TREDPARSE and ExpansionHunter. In this evaluation, GangSTR showed a lower root mean square error (RMSE) rate between true and observed allele lengths for all tested repeats. The authors demonstrated that GangSTR had an advantage over ExpansionHunter and TREADPARSE, especially in alleles that were close to the read length or longer. In addition, GangSTR and ExpansionHunter improved significantly with higher coverage and longer read length.\n\nGangSTR genotyping for disease causing alleles was also tested on validated 14 Huntington's Disease and 25 Fragile X Syndrome real PCR-free WGS data and they reported an RMSE (7.9 and 29.3, respectively) that was lower than for TREDPARSE (8.3 and 34.8, respectively) and ExpansionHunter (10.1 and 27.3, respectively). In evaluations of genotyping a WGS trio, GangSTR was found to have similar performance to HipSTR for shorter alleles (Mousavi et al., 2019).\n\nHipSTR. HipSTR (Willems et al., 2017) is a haplotype-based method for genotyping, haplotyping and phasing STRs. While other STR tools are made for finding true length of repeats independently along the genome, HipSTR takes into account the whole repeat structure on the allele, which may also have missing data. HipSTR accuracy was tested by comparing calls from 118 PCR− WGS samples to capillary electrophoresis data, reporting about 98.8% consistency between the two datasets (Willems et al., 2017).\n\nGATK HaplotypeCaller. GATK HaplotypeCaller (GATK-HC) (McKenna et al., 2010) can also be used for finding SNPs and indels in repeat regions, but it is not specifically made for STR analysis. It has been widely documented that indel calling is not as accurate as SNP calling and indel callers are not ideal for identifying STR mutation due to the lack of reporting repeat genotypes. Instead, indel callers report insertions or deletions of bases relative to the reference, which may or may not be a multiple of the repeat unit, as well as including SNP differences. Dedicated STR callers, however, use information about the repeat unit, composition and repeat length in order to make more accurate genotype calls (Highnam et al., 2013).\n\n\nResults\n\nIn order to evaluate the accuracy and performance of STR genotyping methods, we used a novel evaluation approach applied to exome sequencing data of more than 430 individuals. Several previous comparisons determined accuracy by comparing the estimated lengths of repeats to a known truth, determined from either simulations or alternative assays such as PCR. Here, we took only male individuals and looked at the heterozygosity of the calls only on the X chromosome. As there is only one X chromosome in males, a method that reported only homozygous calls was defined to be more accurate than those that reported heterozygous calls.\n\nWe began with a dataset of 472 males from the Simons Simplex Collection. We had to remove 39 samples for a variety of reasons: six samples were not sequenced with a paired end approach, three samples had no coverage on the Y chromosome so were assumed to be females mislabelled as males, 28 samples produced an error in GangSTR and two samples could not be aligned to the reference genome by BWA due to a software error. The remaining 433 samples (Supplementary Table 1, see Underlying data) (Halman, 2020a) were analysed with LobSTR (Gymrek et al., 2012), RepeatSeq (Highnam et al., 2013), HipSTR (Willems et al., 2017) and GangSTR (Mousavi et al., 2019) (see Methods). In addition, variant calling was performed using the GATK best practices pipeline.\n\nIn brief, the FASTQ files were mapped using BWA-MEM (Li, 2013) and the same BAM files were used as the starting point for running each STR calling method. Each method requires a set of intervals that define repeats to be genotyped. To generate this, we used Tandem Repeats Finder (Benson, 1999) to locate tandem repeats in the hg19 reference genome and detected 224774 STR loci in the X chromosome. Because we are using exome data, we only analysed calls in the 6860 capture regions on the X chromosome. In total, we found 2322 STR loci overlapping the capture regions (Figure 1A), where almost 60% of loci consist of 6 bp repeat units (Figure 1B). In our full data set, across the 433 individuals, we have over a million STR loci for analysis.\n\n(A) Number of STR loci defined by TRF and number of regions in the capture regions of the X chromosome. Overlapping regions include all STR loci that are completely or partially overlapping the target region. Total number of STRs found in target regions is 2322. (B) Distribution of all repeat unit sizes in overlapping regions (2322). STR, short tandem repeat; TRF, Tandem Repeats Finder.\n\nFirst, we looked the ability of a method to make a call at any given locus in the capture regions. By looking at the total number of calls on the X chromosome for each method, we found that LobSTR reported the highest number of loci (Figure 2A). However, the number reported for each individual was variable. Figure 2B shows the distribution of the number of reported loci per individual, with the highest median number of calls by LobSTR (2015), outperforming GangSTR (1967), RepeatSeq (1847) and HipSTR (1834). GATK-HC reported a median of 11 loci per individual but, rather than genotyping all loci, GATK only makes a report when it deems there is a difference from the reference genome. Out of the 2322 loci we investigated, there were 23 loci for which the reference STR length was longer than the read length, but in closer examination none of the tools reported alleles in these loci that were over the read length. However, when looking only at the allele lengths then GangSTR reported 22 unique loci over all samples where at least one of the alleles were longer than the read length, whereas LobSTR reported five and both HipSTR and RepeatSeq four loci.\n\n(A) Total number of calls on the X chromosome over all 433 samples made by each tool. (B) Number of calls made per sample by each tool out of a possible 2322.\n\nGATK only makes calls at positions where there is evidence that the allele is different from the reference. In this dataset, GATK made calls in a total of 346 (14.9%) different loci and 21.4% of these were heterozygous, giving a minimum overall heterozygous rate of 3.1%, assuming all uncalled positions are homozygous. No call either means the allele is a homozygous reference or there is not enough data to make a call. This is one reason why specialised STR callers are better suited for genotyping STR loci.\n\nTo determine the genotyping accuracy of the four specialised STR callers, we first looked at the overall percentage of heterozygous calls to estimate the error rate for each method. Overall, RepeatSeq had the lowest median error rate, with 8675 (1.09%) of its calls being heterozygous. Next was HipSTR with 19459 (2.23%), LobSTR with 27410 (2.96%) and GangSTR with 33204 (3.29%). Again, error rates were variable across individual samples, ranging between 0% and 47.3%. RepeatSeq, HipSTR and LobSTR are generally consistent, with three sample outliers with respect to error rate, while GangSTR has higher variability in error rates across samples (Supplementary Figure 2, Extended data) (Halman, 2020a). Interestingly, for these three samples, the heterozygous percentage increased for LobSTR after the strict filtering. All tools except RepeatSeq recommend filtering the outputs based on quality metrics of the calls (see Methods for details on filtering parameters that were used). Once the recommended filters are applied, we found that the performance of GangSTR improved by 2.76% to 0.53%, HipSTR by 2.14% to 0.09% and LobSTR by 1.32% to 1.64% (Figure 3). However, the median number of calls per sample dropped for LobSTR by 201, HipSTR by 1462 loci and GangSTR by 1512.\n\n(A) Percentage of heterozygous calls over all samples (each dot is a sample) - no filters applied. (B) Percentage of heterozygous calls over all samples after applied the strictest recommended filters for the tools. Since no filters were recommended for RepeatSeq then it has the same values on both plots.\n\nThe recommended filters for each tool were different (for instance, minimum coverage of 100 for HipSTR and 50 for GangSTR) and therefore we next decided to analyse the effect of these filtering parameters separately.\n\nWe investigated the number of heterozygous calls as a function of the repeat unit length, ranging from 1 to 6 bp. We found that all tools exhibit high error rates for 1 bp repeats, which is not surprising as it is difficult to genotype homopolymers due to higher rates of polymerase slippage events. More surprisingly, 2 bp repeats were poorly genotyped by HipSTR, LobSTR and GangSTR and the best results were obtained with RepeatSeq. All other repeat unit lengths produced much more accurate genotypes.\n\nTo investigate the effect of coverage and quality scores on results, we applied call-level filters to our data according to developers' recommendations to get two different datasets: low filtering, where we included all suggested filters except coverage and quality scores, and strict filters, where we also included filters for coverage and quality scores. Then, we looked at the effects of coverage and plotted the error rate as well as percentage of the remaining number of calls as a function of the minimum number of reads supporting the call. We expected the error rate to drop as coverage increased and this was the case for 3, 4, 5 and 6 bp repeat units. However, mono- and dinucleotide repeats did not follow a consistent pattern and the pattern was different between tools. While dinucleotide repeats showed a trend towards a lower error rate with increasing coverage for RepeatSeq and LobSTR, the trend was reversed for GangSTR (Figure 4).\n\n(A) GangSTR, (B) HipSTR, (C) LobSTR, (D) RepeatSeq. Solid line shows the percentage of heterozygous calls as a function of minimum number of reads. Dotted line represents the percentage of remaining calls as a function of minimum number of reads. Y-axis limited to 500 reads. Heterozygous calls are represented in percentages, but the total number of calls is different for each tool, where 100% is 796775 calls for GangSTR, 757432 calls for HipSTR, 848252 calls for LobSTR and 775030 calls for RepeatSeq.\n\nTwo call-level filters are recommended for GangSTR: level 1 filters, which require filtering out calls that have less than 20 reads, and level 2 filters, which require at least 50 reads to support a call. Filtering out all calls with coverage of less than 20 reads brings the heterozygous error rate for the 3–6 bp repeats to between 1.6 and 3.1% at this minimum coverage. Filtering out calls with less than 50 reads improves the error rate even further to 0.83–1.97%. However, by filtering out calls with coverage of less than 20, we lose on average 39.4% of 2–6 bp repeats data, with a median of 1120 loci reported per sample. By filtering out calls with coverage less than 50, we lose on average 71.7% of 2–6 bp repeats data and have a median of 464 loci reported. Interestingly, we see an increase in error rate as a function of coverage when genotyping 2 bp STRs (Figure 4A). Unlike the other tools, where heterozygous percentage decreases when coverage increases and remains relatively steady, GangSTR is not so consistent and fluctuates around 1%.\n\nHipSTR excludes calls that have coverage less than 100 by default unless specified otherwise. This coverage gives high accuracy but also filters out 75.9% of data. We investigated reducing the minimum coverage and found HipSTR has excellent accuracy even with minimum coverage of 25: 0.02–0.04% heterozygous rate for 3, 5 and 6 bp and 0.16% for 4 bp repeats (Figure 4B). In addition, a minimum coverage of 50 improves results even more and the maximum error rate for these repeats is 0.01% at this minimum coverage (median of 899 calls per sample). HipSTR also struggles to genotype 2 bp repeats, having a heterozygous error rate around 5.2% for coverage of both 25 and 50. Therefore, by decreasing the filtering parameters for coverage to only exclude the calls less than 25x coverage, we retain on average 76.6% of data for 2–6 bp repeat units, with a moderate call rate (median of 1369 calls per sample).\n\nLobSTR recommends filtering out calls with coverage less than five, which gives us a heterozygous percentage for 3–6 bp repeat units at this minimum coverage of 0.44–0.90% per repeat unit length. This filters out on average 10.0% of calls for 2–6 bp repeats, giving a median of 1840 reported alleles per sample. The accuracy for these repeats increases as a function of coverage and considering the amount of calls that are filtered out, a minimum coverage of 5–10 might be the best compromise. By filtering out calls with coverage less than 10, the heterozygous error rate at this minimum coverage is 0.55% for 4 bp and 5 bp repeats, 0.48% for 3 bp repeats and 0.36% for 6 bp repeats, while still retaining 74.5% of calls on average for 2–6 bp repeats (median of 1555 calls per sample). Increasing the minimum coverage to 20 reads would improve our results by a further 0.2% but also filter out significantly more calls. For dinucleotide repeats, accuracy seems to get better with higher coverage and reaches 1% error rate when the minimum coverage is 33; however, the error rate increases again when coverage increases over 42 (Figure 4C).\n\nAuthors of RepeatSeq do not recommend any additional filtering and we found high accuracy even at low coverage. Filtering out all calls less than coverage of five will result in an error rate for 3–6 bp repeat units of 0.04–0.08% and only an average 7.1% of 2–6 bp repeat calls filtered out, leaving an average of 1728 reported calls per sample. Among all tools, RepeatSeq shows the best accuracy for dinucleotides, having an error rate no more than 1.81% (Figure 4D).\n\nAs mentioned, we saw unusually high error rates for dinucleotide repeats in nearly all tools, so we examined these repeats in more detail. Curiously, we found that for GangSTR and LobSTR, the unusually high error rate of 2 bp repeats were due to AC/TG repeats, while other repeat units do not exhibit these characteristics (Figure 5). The same pattern was observed for RepeatSeq but at lower error rates. This information is not easily available for HipSTR as it does not report the repeat unit.\n\nPercentage of heterozygous calls per minimum number of reads for dinucleotide repeats for (A) GangSTR, (B) LobSTR and (C) RepeatSeq, divided by the three possible repeat unit sequences.\n\nBesides coverage, the second parameter which is commonly used for filtering in all tools is the call quality or quality score produced by each algorithm. We next investigated the effects of quality scores on accuracy by relaxing the coverage filtering and looking at the quality scores in different bins across the score range (Figure 6). GangSTR's level 2 filters recommend filtering out calls that have quality scores below 0.9. We see in Figure 6A that the heterozygous error rate fluctuates at lower scores and starts to decrease from quality scores above 0.6 for all repeats except for homopolymers. We reach a 1% heterozygous error rate when excluding calls that have quality scores below 0.81 for 3 bp repeat units, 0.66 for 4 bp units, 0.77 for 5 bp units and 0.93 for 6 bp repeat units. We see the lowest heterozygous error rate at the quality score of 1.0, but we also determined a sharp drop in the number of reported genotypes after excluding calls with quality scores below 1.0, and we also lose 61.7% of 3–6 bp repeats data on average. The recommended 0.9 seems a reasonable suggestion for balancing the accuracy with how much data we will have left after the filtering. We can also see that the accuracy of 1 bp repeat calls improves with the highest quality score (1.0) and a stronger filter for this repeat unit may be appropriate.\n\n(A) GangSTR, (B) HipSTR, (C) LobSTR, (D) RepeatSeq. Each bin is from marked range (inclusive) to the end value (exclusive), except the last one. Dot size represents the number of calls in that range of the repeat unit length (rows).\n\nHipSTR, similarly to GangSTR, recommends filtering out calls with a quality score less than 0.9. In Figure 6B, we can see that on average the calls with a score below 1.0 have a high heterozygous error rate, and keeping only the ones with the highest quality score will improve overall accuracy. Indeed, 98.5% of calls have a quality score of 1.0 and therefore, we only lose a fraction of data while filtering out calls with a quality score below 1.0, which may be a good trade-off.\n\nLobSTR recommends filtering out calls with a call quality score less than 0.8. As seen in Figure 6C, the heterozygous error rate fluctuates but generally shows higher accuracy with higher quality scores for 1–3 bp repeats, while for 4–6 bp there is no clear trend below a quality score of 0.9. There is no significant improvement in overall accuracy when we remove calls with quality scores less than 0.8, or even 0.9, which might be due to the fact that, similar to HipSTR, the majority of calls (94.3%) have a quality score of 1.0. We do see improvement when only leaving calls with quality of 1.0, particularly in 1–2 bp repeat units, which shows utility in filtering out the least accurate calls and since the majority of data has a quality score of 1.0, this filtering could be a good choice, as also suggested for HipSTR (see Supplementary Figure 3, Extended data) (Halman, 2020a).\n\nRepeatSeq does not recommend any filtering and reports quality scores on a Phred scale (Figure 6D). We determined that filtering out calls with Phred quality score of 10 or less improves the accuracy of all repeat units. Accuracy of genotyping mono- and dinucleotide repeats continues to improve as a function of Phred scores, while the best accuracy is observed at the highest quality score. On the flip side, the number of calls also decreases, and at the highest Phred scores, we are left with 31.6% of homopolymers and 87.8% of dinucleotide repeats. However, it only filters out 1.2% of 3–6 bp repeats data. Overall, filtering data based on the quality scores may be reasonable if looking at mono- or dinucleotide repeats and accuracy is an important factor.\n\nLobSTR, HipSTR and RepeatSeq use types of reads where the STR region has to be completely in the read. However, GangSTR uses more classes of reads that may give rise to false positives. Therefore, to make a more direct comparison, we decided to look at GangSTR results where we filtered out all other classes of reads besides the enclosing ones, marked here as GangSTR (enc.). Compared to the previous GangSTR results, we now see lower error rates for all repeat units as well as no substantial fluctuation in higher coverage that was apparent previously in Figure 4A. At a coverage of 20, GangSTR (enc.) has a heterozygous error rate of 0.53–0.99% for 3–6 bp repeats, while 58.6% of 2–6 bp repeats data is filtered out, with a median of 699 calls per sample (Figure 7A). When we increase the minimum number of reads to 50, we can see even further improvement. This results in an error rate of 0.01–0.27% for 3–6 bp repeats; however, this also filters out 86.3% of 2–6 bp repeats data (median of 152 calls per sample).\n\nPercentage of (A) heterozygous calls per minimum number of reads and (B) in different quality score ranges for GangSTR (enc.). Solid line on (A) represents the percentage of heterozygous calls per minimum number of reads and dotted line represents the percentage of remaining calls per minimum number of reads. Dot size on (B) represents the number of calls in that range of the repeat unit length (rows) and each bin is from marked range (inclusive) to the end value (exclusive), except the last one.\n\nWe also looked at the dinucleotides separately (see Supplementary Figure 4, Extended data) (Halman, 2020a) and found that it is considerably lower compared to the results of GangSTR. However, we determined that AC/TG repeats still have a higher error rate compared to other repeat units (Figure 4A and Figure 7A). Results of quality scores are quite similar to GangSTR's, where we see improvements at high quality scores (Figure 7B).\n\nFinally, we benchmarked all tools to determine their average running time. We ran each tool genome wide by using one, two, four and eight cores and determined that only RepeatSeq supports multithreading, which allows the tool to run faster when utilising more processor cores. In particular, we saw that for samples that have coverage on target regions of around 90x, the average running time on one core for LobSTR was 9 min 31 sec, giving it a clear advantage compared with HipSTR at 1 h 22 min, RepeatSeq 2 h 58 min and GangSTR at 3 h 4 min. By utilizing more cores, LobSTR's, GangSTR's and HipSTR's running time remained the same, while RepeatSeq's time improved.\n\n\nDiscussion\n\nWe used a novel way of evaluating the error in short tandem repeats genotyping methods where we analysed STR calls on the X chromosome of male samples. Because this is a hemizygous chromosome, we determined a relative error rate as the rate of heterozygous genotypes. We performed this evaluation on a human exome dataset of 433 samples, resulting in the evaluation of more than a million STR loci. Exome sequencing is widely used, but the PCR step in library preparation causes a challenge for STR genotyping tools due to the interference of stutter noise. This is the first independent evaluation of these STR genotyping tools that we are aware of.\n\nMany of the tools do not claim to be able to accurately genotype homopolymers and we found that indeed all tools had difficulty with these repeats, resulting in a high error rate. There was also no clear correlation between minimum coverage and accuracy of genotyping homopolymers, but using the highest quality scores did improve the accuracy. Interestingly, most tools produced high error rates for genotyping dinucleotides as well, which we later found to be mainly caused by AC/TG repeat units. One who analyses dinucleotide repeats with these tools should be aware of the differences in accuracy of genotyping different repeat units and carefully interpret the results of AC/TG repeat units. Repeat units with a length of 3–6 bp were all relatively accurate and similar across tools, with only minor differences. However, genotyping was slightly less accurate for 3 bp length repeats in low coverage and low quality scores, but differences were reduced with proper filtering. We found that LobSTR was able to report the highest number of genotypes at a heterozygous error rate of less than 1%.\n\nThere are certain filtering parameters suggested for each tool and we examined the effects of coverage and quality scores across all tools. However, some tools have further parameters that could be explored that were not part of our investigation. In general, we found that higher quality scores increased the accuracy of results at the cost of losing some potentially accurate calls. The relationship with coverage was more complex but some coverage filtering improved results for all tools. Which parameters to use depends on the aim of the analysis. For example, more calls may be desirable to begin a screen, or more accuracy may be desirable if selecting potential disease associated loci. When one does an exploratory analysis to find potential loci of interest that can be followed up with alternative methods, then lowering filtering parameters for coverage and quality scores for certain tools could be a good approach as it leaves us with larger portion of data. We found that even in exome data, we can use these tools to genotype tens of thousands of loci.\n\nUnlike the other tools we used in this analysis, GangSTR utilises four different types of reads, which can help to pick up the locus other tools cannot (such as those longer than the read length). However, these can also produce genotyping errors. In our analysis, we first looked at GangSTR results that included all four classes of reads and then we excluded all calls where only spanning or bounding class reads were present, as suggested by the tool authors. This filtering increased the genotyping accuracy of the tool (we also looked at the results where we skipped this filtering parameter but this did not improve results). Still, compared to other tools, GangSTR showed a higher error rate. Finally, we decided to look only at the enclosing class of reads as the other tools do and determined an error rate around three times lower at 20x and bigger gains at higher coverage. On the other hand, that change will result in losing the ability to genotype alleles longer than the read length, which is GangSTR's important addition. We also found that HipSTR has a very high accuracy for 3–6 bp repeats when coverage is at least 50x. Excellent accuracy was also found for RepeatSeq at very low coverage and this was the most accurate among the tools for genotyping dinucleotides. In addition, RepeatSeq is the only tool that supports multithreading and therefore can run faster by allocating more cores.\n\nHere, we have presented one way of performing an evaluation and this approach does not look at accuracy of the estimated allele length, which is a limitation of the study. In addition, it is difficult to rule out a bias towards tools that default to genotyping an allele as a homozygous reference by the software. Our comparisons were specifically analysing an exome dataset that was PCR amplified, where a tools' noise model may play an important role. Therefore, tools may perform differently when we analyse PCR-free WGS datasets.\n\nIn conclusion, all these tools are built to genotype STRs but have different strengths and weaknesses. Based on our analysis there is no clear overall winner. RepeatSeq and HipSTR are the best when considering genotyping error rate even with low coverage. On the other hand, GangSTR has an advantage because it is the only tool among them that can call alleles longer than the read length but shows a higher error rate, unless looking at only the enclosed class of reads, which in turn would lose the GangSTR's advantage of picking up long genotypes. In addition, GangSTR is the newest tool and so comes with reference files for different reference builds that are periodically updated according to the tool's webpage. The correct choice of a tool and the subsequent filtering depends on the aim of the analysis, and might be influenced by available hardware resources and time limit for running tools.\n\n\nMethods\n\nIn order to compare all STR tools, we ran each one of them on the same dataset. We used the data from the publicly available Simons Simplex Collection (SSC) for our analysis.\n\nIn total there were 238 families where only males were selected for our analysis to avoid heterozygous sites in the X chromosome, assuming that any multiallelic STR calls should be a result of PCR and/or sequencing errors. Male samples were determined by using metadata of samples (472 samples) and quality controlled by looking at the coverage on X and Y chromosomes. Results of the analysis led to the exclusion of three samples as they had no coverage on the Y chromosome. Out of the remaining 469 samples, we excluded six single-end read sequenced files as well as 28 paired-end read sequenced samples that did not work on GangSTR, and two additional samples that had issues with mapping, which left us with 433 samples in total (Supplementary Table 1, see Underlying data) (Halman, 2020a).\n\nGenomic DNA of the final (433) samples used in this analysis was extracted from whole blood, exomes were captured with NimbleGen EZ Exome v2.0 (Roche Nimblegen, Inc., Madison, WI) reagents and sequenced using Illumina (San Diego, CA) GAIIx (N = 271) or HiSeq 2000 (N = 162) at the Yale University School of Medicine.\n\nAll computational steps (tools and parameters used) are described in this section.\n\nAll whole-exome sequencing files in Simons Simplex families were downloaded from NCBI Sequence Read Archive (SRA) and converted to FASTQ files by using fastq-dump:\n\n\n\nThe human reference sequence hg19 (February 2009 assembly) was downloaded from UCSC and the \"hg19.fa\" file was created by and indexed using Samtools v1.10 (Li et al., 2009):\n\n\n\nCreating FASTA sequence dictionary file for GATK analysis:\n\n\n\nTandem Repeats Finder v4.09 (Benson, 1999) was used to find STRs (1–6 bp repeat unit length) in the hg19 reference genome using the following command and parameters:\n\n\n\nA custom-made Python script named trf2bed.py (Halman, 2020b) was used to extract data from the TRF output file to generate a BED regions file for LobSTR, GangSTR, HipSTR, RepeatSeq and GATK.\n\n\n\nReads from FASTQ files were aligned to the hg19 reference genome using BWA-MEM v0.7.17 and aligned BAM files were merged and indexed using Samtools v1.10 (Li et al., 2009).\n\n\n\n\n\nTo follow the best practices of GATK duplicate reads were removed:\n\n\n\nCoverage of BAM files on target regions was found with the MosDepth v0.2.4 tool, followed by calculating the median and average coverage:\n\n\n\n\n\n\n\nGangSTR v2.4 (Mousavi et al., 2019) was executed with the following parameters:\n\n\n\n\n\nStrict filtering was done as recommended by the developer using dumpSTR, which is part of TRTools package:\n\n\n\nSince we were looking the relationship between coverage and quality scores, and genotyping accuracy separately, we did additional filtering (partial filtering), where we discarded the filtering on calls with low coverage or low-quality scores:\n\n\n\nRepeatSeq v0.8.2 (Highnam et al., 2013) was executed with the following parameters:\n\n\n\nLobSTR v4.0.6 (Gymrek et al., 2012) was downloaded and a custom lobSTR reference was made using lobstr_index.py and GetSTRInfo.py scripts as follows:\n\n\n\n\n\nLobSTR’s allelotype was used to call STRs and it was run with default parameters, with and without the the \"--no-rmdup\" flag:\n\n\n\nWillems and colleagues explored the effects of recommended allelotype options for lobSTR (--filter-mapq0, --filter-clipped, --max-repeats-in-ends and --min-read-end-match), but found the optimal settings for lobSTR does not include these parameters and best results are obtained with default ones, which was also reported for RepeatSeq (Willems et al., 2017), and therefore we decided to run both tools with the default parameters.\n\nWe did the strict filtering with the lobSTR's filtering tool, based on the author's recommendations for whole genome data:\n\n\n\nAnd the partial filtering:\n\n\n\nHipSTR v0.6.2 (Willems et al., 2017) was executed with the following parameters:\n\n\n\nStrict filtering was done according to the developer's recommendations:\n\n\n\nSince we ran HipSTR with \"--min-reads 2\" parameter, we additionally filtered out all calls that had less than 100 reads, as this is the default parameter that HipSTR uses.\n\nPartial filtering was done:\n\n\n\nGATK v4.1.2 was executed with the following standard parameters:\n\n\n\nData were analysed using GATK best practice guidelines (DePristo et al., 2011) up to variant calling. Variant calling was performed with the HaplotypeCaller in GATK (Poplin et al., 2017).\n\nA custom-made Python script named extract-data.py (Halman, 2020b) was created to extract data from outputted VCFs of all tools ran:\n\n\n\n\n\nIn particularly, only calls in the X chromosome were extracted out. In case of filtering, only calls that passed the filter were extracted out. The output file contained information about all STR loci found in the VCF file, having the following fields: sample name, locus, chromosome, start and end coordinates of the STR region, motif (repeat unit), length of motif, length of the reference, length of alleles, genotype, number of total reads, number of reads supporting the call in each class and the quality score.\n\nThe data was then analysed in R by using str-analyse.R together with str-analyse.functions.R script (Halman, 2020b). Bioconductor's GenomicRanges package for R (Lawrence et al., 2013) was used to find and then filter out all calls that fell outside of the target regions. All STR regions that were entirely or partially inside of the target region were included in the analysis, however, all duplicate loci were removed. When calculating heterozygous percentage per minimum number of reads or quality score bins, we only included the results when there were minimum of ten results (samples) to use and calculated the percentage again for each repeat unit length after each read or after 1/10 quality score bin.\n\nTo see the performance of the STR specific tools we selected five WES samples that had a median coverage on target regions closest to 90x (between 88.6x and 91.6x) and calculated the time each tool ran on each sample individually by using either one, two, four or eight processor cores on the same server that has Intel(R) Xeon(R) 2.60 GHz processors and maximum of 16 GB RAM. Each test was repeated three times and the average time was calculated. Timing was performed with the UNIX time command.\n\nAn earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.02.03.933002).\n\n\nData availability\n\nThe Simons Simplex Collection dataset, Accession number SRP010920: https://identifiers.org/insdc.sra:SRP010920\n\nHarvard Dataverse: Supplementary information for the \"Accuracy of short tandem repeats genotyping tools in whole exome sequencing data\" article. https://doi.org/10.7910/DVN/RWTGWK (Halman, 2020a)\n\nThis project contains the following underlying data within the ‘Supplementary_information’ PDF:\n\n- Supplementary Table 1 (list of all accession numbers used in the analysis)\n\nHarvard Dataverse: Supplementary information for the \"Accuracy of short tandem repeats genotyping tools in whole exome sequencing data\" article. https://doi.org/10.7910/DVN/RWTGWK (Halman, 2020a)\n\nThis project contains the following extended data within the ‘Supplementary_information’ PDF:\n\n- Supplementary Figure 1 (Coverage of X and Y chromosome of samples marked as male in the metadata)\n\n- Supplementary Figure 2 (Percentage of heterozygous calls over all samples with and without filters applied)\n\n- Supplementary Figure 3 (Percentage of heterozygous calls as a function of minimum quality score)\n\n- Supplementary Figure 4 (Percentage of heterozygous calls per minimum number of reads for dinucleotides)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nSource code available from: https://gitlab.com/andreassh/research-str-wes\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3695971 (Halman, 2020b)\n\nLicense: MIT",
"appendix": "Acknowledgments\n\nThe authors are thankful to Katrina Bell for their comments and suggestions for the article.\n\n\nReferences\n\nBenson G: Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27(2): 573–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBjörn N, Pradhananga S, Sigurgeirsson B, et al.: Comparison of Variant Calls from Whole Genome and Whole Exome Sequencing Data Using Matched Samples. Next Gener Seq Appl. 2018; 5(1). Reference Source\n\nBudiš J, Kucharík M, Ďuriš F, et al.: Dante: genotyping of known complex and expanded short tandem repeats. Bioinformatics. 2019; 35(8): 1310–17. PubMed Abstract | Publisher Full Text\n\nCao MD, Tasker E, Willadsen K, et al.: Inferring short tandem repeat variation from paired-end short reads. Nucleic Acids Res. 2014; 42(3): e16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaspar SM, Dubacher N, Kopps AM, et al.: Clinical sequencing: From raw data to diagnosis with lifetime value. Clin Genet. 2018; 93(3): 508–19. PubMed Abstract | Publisher Full Text\n\nDashnow H, Lek M, Phipson B, et al.: STRetch: Detecting and discovering pathogenic short tandem repeat expansions. Genome Biol. 2018; 19(1): 121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDePristo MA, Banks E, Poplin R, et al.: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011; 43(5): 491–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDolzhenko E, van Vugt JJFA, Shaw RJ, et al.: Detection of long repeat expansions from PCR-free whole-genome sequence data. Genome Res. 2017; 27(11): 1895–903. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDolzhenko E, Deshpande V, Schlesinger F, et al.: ExpansionHunter: a sequence-graph-based tool to analyze variation in short tandem repeat regions. Bioinformatics. edited by Inanc Birol, 2019; 35(22): 4754–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFungtammasan A, Ananda G, Hile SE, et al.: Accurate typing of short tandem repeats from genome-wide sequencing data and its applications. Genome Res. 2015; 25(5): 736–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGymrek M: A genomic view of short tandem repeats. Curr Opin Genet Dev. 2017; 44: 9–16. PubMed Abstract | Publisher Full Text\n\nGymrek M, Golan D, Rosset S, et al.: lobSTR: A short tandem repeat profiler for personal genomes. Genome Res. 2012; 22(6): 1154–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalman A: Supplementary information for the “Accuracy of short tandem repets genotyping tools in whole exome sequencing data” article. [dataset]. Harvard Dataverse, V1. 2020a. http://www.doi.org/10.7910/DVN/RWTGWK\n\nHalman A: Source code for the \"Accuracy of short tandem repeats genotyping tools in whole exome sequencing data\" article. [dataset]. Zenodo. 2020b. http://www.doi.org/10.5281/zenodo.3695971\n\nHannan AJ: Tandem repeats mediating genetic plasticity in health and disease. Nat Rev Genet. 2018; 19(5): 286–98. PubMed Abstract | Publisher Full Text\n\nHighnam G, Franck C, Martin A, et al.: Accurate human microsatellite genotypes from high-throughput resequencing data using informed error profiles. Nucleic Acids Res. 2013; 41(1): e32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKozarewa I, Ning Z, Quail MA, et al.: Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods. 2009; 6(4): 291–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKristmundsdóttir S, Sigurpálsdóttir BD, Kehr B, et al.: popSTR: population-scale detection of STR variants. Bioinformatics. 2017; 33(24): 4041–48. PubMed Abstract | Publisher Full Text\n\nLawrence M, Huber W, Pagès H, et al.: Software for Computing and Annotating Genomic Ranges. PLoS Comput Biol. 2013; 9(8): e1003118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H: Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. 2013; 1–3. Reference Source\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMousavi N, Shleizer-Burko S, Yanicky R, et al.: Profiling the genome-wide landscape of tandem repeat expansions. Nucleic Acids Res. 2019; 47(15): e90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoplin R, Ruano-Rubio V, DePristo MA, et al.: Scaling accurate genetic variant discovery to tens of thousands of samples. BioRxiv. 2017; 201178. Publisher Full Text\n\nTang H, Kirkness EF, Lippert C, et al.: Profiling of Short-Tandem-Repeat Disease Alleles in 12,632 Human Whole Genomes. Am J Hum Genet. 2017; 101(5): 700–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang H, Nzabarushimana E: STRScan: targeted profiling of short tandem repeats in whole-genome sequencing data. BMC Bioinformatics. 2017; 18(Suppl 11): 398. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTankard RM, Bennett MF, Degorski P, et al.: Detecting Expansions of Tandem Repeats in Cohorts Sequenced with Short-Read Sequencing Data. Am J Hum Genet. 2018; 103(6): 858–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWillems T, Gymrek M, Highnam G, et al.: The landscape of human STR variation. Genome Res. 2014; 24(11): 1894–904. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWillems T, Zielinski D, Yuan J, et al.: Genome-wide profiling of heritable and de novo STR variations. Nat Methods. 2017; 14(6): 590–92. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "61590",
"date": "26 May 2020",
"name": "Elizabeth A. Worthey",
"expertise": [
"Reviewer Expertise Computational biology. Genetics."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article compares a number of tools released to characterize the presence and nature of repetitive elements from genomic data. The stated goal of the analysis is to generate findings that would help other researchers choose suitable tools and parameters when performing this type of analysis in their own lab. Aspects considered included the ability to call as well as accurately genotype repetitive elements. The analysis is not limited to long disease associated repeats, which has often been the case for obvious reasons.\nThe authors compared four different short tandem repeats genotyping tools making use of whole exome sequencing data. They sought to determine genotyping performance and limits. They also examined parameters settings to determine if and how to refine the analysis to increase accuracy. The authors made use of a readily accessible dataset for these analyses.\nAs would be expected, they found that all tools showed reasonable performance when genotyping repeats of 3-6 bp in length. As would be expected, they found that accurate homopolymer genotyping was challenging for all tools, with a high error rate being seen cross the board. They found issues nin mono and dinucleotide repeats as would also be expected/has been shown previously. The authors categorized tools based on a number of factors. For example they found that LobSTR made the most calls (which may or may not be a good thing) and was the fastest tool, while RepeatSeq and HipSTR exhibited the lowest heterozygous error rate at low coverage. The methods, tools reviewed, and dataset used for testing were clearly presented. The reasoning for selecting those tools was also well laid out. The article cited the current literature in the field. The study design was appropriate for the purpose of comparing tools to define specific strengths and weaknesses. It might have been nice to have more of an in depth discussion as to how the various tools differed in terms of their algorithm and how this related to the results and conclusions made. The authors stated that a goal of the analysis would be to generate a framework useful for others looking to compare and contrast tools to support selection for research. As such it might have been nice to include some specifics on ease of installation, levels of support etc. for the various tools etc. Maybe they were all very easy and well supported in that regard.\nThe authors provide specific details of the parameters used to run each tool. The majority appear to be run using defaults; there does not seem to have been a huge amount of parameter refinement . This may be due to limitations in what is possible with these tools. The authors provided links to the datasets used for the comparison. The source data used is not provided per se because it is access controlled. The data is, however, a well known publicly available dataset and it should not be very difficult for others to obtain access. People wishing to recapitulate the study would simple have to apply. The authors provided links to the code they used. All combined; these aspects should make for ease of replication by those interested.\nVery minimal statistical analysis and its interpretation was required because of the nature of the analysis presented, but zenodo links to scripts were provided. The conclusions drawn were adequately supported by the findings that they presented. Testing of the X chromosome for accuracy in variant calling is not a novel approach. Study of the X to explore the genotype and location of repetitive sequences is also not a novel approach. Combining the two approaches to use the X chromosome to specifically examine repetitive sequences has not been published.\nThe study is essentially a comparison of tools. As such, it is well organized and executed and would be of interest for people looking to set up pipelines in their lab. As the authors acknowledge most findings gleaned from their analysis were not surprising; they primarily confirmed known constraints or issues with the tools and their analysis confirmed existing knowledge regarding the nature of the repetitive sequences studied. Selection of the X chromosome was a nice way to undertake analysis that would be likely to be adopted by others. Given this background, this work would seem to have value primarily for education purposes to help researchers select and set up tools. It would be useful for that purpose. It does not present much, if anything, in the way of novel generalizable research knowledge on the repetitive nature of the human genome.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "63745",
"date": "17 Jun 2020",
"name": "Jan Radvanszky",
"expertise": [
"Reviewer Expertise JR - human molecular genetics",
"genomic technologies",
"JB - bioinformatics",
"computational biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAndreas Halman and Alicia Oshlack prepared and submitted a very interesting manuscript dealing with the results of their comparison of four selected STR genotyping tools (LobSTR, HipSTR, GangSTR and RepeatSeq), which are designed to process data from massively parallel sequencing (here used for exome sequencing data sets). More precisely, they compared five tools in the initial phases of the experiments, however, GATK HaplotypeCaller is not a dedicated STR tool and was disregarded in later phases of the study because of its several limitations. Although the study is well designed, well evaluated and also well written, it has one substantial limitation - the study design itself. The authors chose an elegant and low-cost, but still high-throughput approach that relies on the biological fact that healthy males (at least in humans and other mammals) bear one X chromosome in their genome, so they are hemyzygous for the major part of this chromosome (when not considering the pseudoautosomal regions). Based on this phenomenon, they “expect(ed) all calls (coming from the X chromosome) to be homozygous” therefore they evaluated the proportion of “incorrect calls revealed by a heterozygous call” to determine the “accuracy of genotyping”. Genotyping accuracy, however, has several aspects and the number of incorrectly called heterozygous positions (i.e. the false heterozygous rate) is only one part of it. The approach chosen by the authors, therefore, does not allow to determine the total genotyping accuracy, only the rate of false heterozygous calls, i.e. the ability of tools to discriminate between heterozygous and homozygous state. We need to mention here, that this limitation is clear from the study and is properly acknowledged by the authors in several places in the manuscript (both in the Introduction and also in the Discussion). We would like to emphasize, therefore, that we do not consider this limitation for an insufficiency of the study design. On the other hand, because of the inadequate wording chosen by the authors (genotyping accuracy) we consider the conclusions described by these words only partly supported.\n\nWe agree with the authors, however, that they present a “different but complementary approach” to evaluate the performance of STR genotyping tools, at least with respect to their ability to make a call, and to correctly assign homozygous calls to homozygous STR positions. We are sure that this approach will become a well established approach not only in future research studies, but also in everyday practice. Following thorough review of the manuscript, we would like to suggest some major and several minor points to be considered by the authors:\n\nMajor suggestions:\nWe would like to suggest rewording of “genotyping accuracy” (and similar formulations, such as “accuracy of genotyping”, “performance of genotyping tools”, etc.) with a more appropriate formulation - throughout the manuscript, everywhere, where they are used with respect to the results of the authors evaluation. Maybe with “false heterozygous rate”?\n\nPage 10; Part “Effects of repeat unit and quality scores on accuracy” - the conclusions, according to which:\n\nIn case of GangSTR “We can also see that the accuracy of 1 bp repeat calls improves with the highest quality score (1.0) and a stronger filter for this repeat unit may be appropriate”, seems not to be correct, since there is a relatively wide range of lower quality scores in which GangSTR leads to lower false heterozygous rates (and also with larger dot sizes) - at least according to Figure 6a For HipSTR and 1 bp repeats there is a similar exception from the general trend that “keeping only the ones with the highest quality score will improve overall accuracy”\n\nFigure 5 and results for the dinucleotide repeats:\n\nWhy is the GC/CG dinucleotide missing from the compared possible dinucleotide sequences? If it is by mistake, it would be useful to add this dinucleotide to the comparison. If it has a specific reason, this should be discussed in the text. Similarly to dinucleotides, there were also higher error rates found for homopolymers. It would be interesting to try to evaluate the error rate also for each of the four possible homopolymers and to see whether there are different trends between them (like the marked AC/TG trend with GangSTR and LobSTR).\n\nMinor suggestions:\nPage 3; First indent of Introduction: with regard to the standard methods to genotype STRs it would be better to change “gel electrophoresis” to “capillary electrophoresis”, since gel electrophoresis most commonly refers to agarose or polyacrylamide gels\n\nPage 6; Third and fourth indents of the part called “Dataset”: We would suggest to merge the two parts dealing with GATK (“GATK-HC reported a median...” and “GATK only makes calls at positions...”) into one, because they are now unnecessarily separated by the part dealing with the “...23 loci for which the reference STR length was longer…”\n\nFigure 2.: We recommend to:\n\nUse the same order of tools in the part A) and B) of the figure Include GATK in the charts, since GATK was also evaluated and compared in this step of the study (even if it resulted in small numbers of calls and its numbers does not necessarily reflect its ability to call genotypes, since it calls only alleles which are different from the reference genome). Although this will most probably lead to “simple rows” in the lower parts of the chart, because of the significantly lower numbers obtained with GATK, we believe that such inclusion will strengthen the visual recognition of the significant differences between the tools We believe also that adding the respective numerical values for each tool (for example up to the top of each bar in the bar chart, and under the median in the box-plot) can further enhance the informational value of the charts Provide also comparison of number of calls after applying the optimal filters (mentioned at the end of the “Dataset” section)\n\nPage 7; Part “Effects of repeat unit and coverage on accuracy”:\n\nWith few exceptions, nearly each experiment of the study is visualized through plots. We think that the number of heterozygous calls as a function of repeat unit length for each compared tool would deserve a plot too, since we believe that readers would appreciate the visualisation of these results\n\nWe think that the last sentence on the page (“While dinucleotide repeats showed a trend towards a lower error rate with increasing coverage for RepeatSeq and LobSTR, the trend was reversed for GangSTR”) does not describe accurately the complex trends visualised by the plots on Fig.4 (for LobSTR and HipSTR). LobSTR for example shows a lowering trend only up to a certain point, where it sharply increases. This seems to be, moreover, an opposite trend to that seen for HipSTR, which has a rather increasing error rate (although with little fluctuations) up to a certain point when it suddenly drops Do the authors have an explanation for the sudden change in trends encountered in the LobSTR and HipSTR results for mono- and dinucleotide repeats?\n\nFigure 4. (and other line plots): The lines in the plots are difficult to read, especially in a printed form. We suggest reworking of the graphs for better clarity, for example with thicker trend lines, white background instead of grey, etc.\n\nFigure 6: Can the authors include an explanation (or at least a hypothetical reason) to the “strange” trend encountered in the HipSTR results?\n\nPage 13; The very last sentence of the “Results” section: We recommend to specify the respective time also for RepeatSeq, determined after the utilisation of more cores (while RepeatSeq´s time improved to ....)\n\nAlthough they comprise only a relatively small part of X chromosome, the pseudoautosomal regions (PAR1 and PAR2) should be mentioned in the study. Together with the information, whether these regions were excluded or included in the target regions for the present study - alternatively, whether any of the called STR loci mapped to these regions (and if yes, then their proportion to the remaining region should be mentioned)\n\nPage 13; “Discussion”: We suggest to move the actual fifth indent (“Here, we have presented one way of performing...”) after the first one (“We used a novel way...”), because together they will form a more consistent “introduction” to Discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "63748",
"date": "30 Jun 2020",
"name": "Maria Anisimova",
"expertise": [
"Reviewer Expertise computational molecular evolution",
"bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn recent years short tandem repeats (STRs) drew increased attention because of their frequent associations with disease, as well as their abundance and a diversity of roles in protein functions. Consequently, many methods were developed for detecting and genotyping tandem repeats. With wide variation in methodology, and its related settings, it is often hard to know which software can provide an optimal performance and be integrated in bioinformatics pipelines. The study by Halman and Oshlack “Accuracy of short tandem repeats genotyping tools in whole exome sequencing data” contributes some insights for scientists who wish to study short tandem repeats (STRs) loci in genomic data. The article is clearly written and should be practically useful for those developing relevant bioinformatics pipelines.\n\nThe authors evaluate four STR genotyping tools, they provide a clear description and reasoning for their choices and point out the main differences between the tools. Overall, the results are not surprising and could be expected: the evaluated tools all have different strengths and weaknesses and the choice should be driven by the specific goals of the analysis. Clearly, different filtering settings will produce results of different accuracy, with a trade-off between the genotyping accuracy and the highest number of calls. As expected, homorepeats and dinucleotide repeats are the most problematic and there is currently no tool providing a good solution for such STRs. Apart from the valuable main message, the quantitative results should not be generalized, these are more to get a “feeling” of the expected accuracy than any guarantee. Indeed, it is difficult to directly compare methods that differ in so many ways: conceptually, by assumptions of random noise model, alignment procedures, filtering options and cut-offs, etc. Nevertheless, I applaud the authors for trying to accomplish (at least partially) this difficult task.\n\nAt the same time, I would like to raise some points for reflection. Note, that all evaluated tools required a set of defined STRs loci. These were detected using the TRF tool (Benson 1999)1 based on self-alignment. In our article Schaper et al. (2012)2, we have evaluated several tandem repeat (TR) predictors, including TRF. As one can expect (and similar to the findings in the article reviewed above), due to the heterogeneity of the methods in terms of their main concepts and assumptions, we observed a wide spectrum of variable performances over the space of TRs. Specifically, for TRF, we observed that this predictor is very conservative – having a low false positive rate across the TR space (in terms of TR unit numbers and unit length) but also a very low power (high false negative rate). For repeats with very short units, TRF is extremely conservative. Therefore, such bias in STR detection by TRF may have consequences on the observed by the authors distribution of STR unit sizes (e.g., in Figure 1b, the overrepresentation of longer 6-nt units may be explained simply by the TRF-specific bias).\nTherefore, I would be curious how the results of Halman and Oshlack would change, both relatively and quantitatively, were they to use a TR annotation tool with contrasting properties, i.e. that is better at detecting STRs specifically. For example, this could be Xtreme (Newman and Cooper, 2007)3 or a meta TR predictor, each followed by statistical filtering (as we suggest in Schaper et al 20122 and also in Anisimova et al. 2015)4. These approaches typically suggest a TR unit distribution dominated by homo- and dinucleotide repeats (eg, see Delucchi et al. 20205).\nFinally, the authors also mention the current gap in methodology for indel calling. For repeat regions indels become particularly important - as a consequence of TR unit number variation, but also due to regular indel events. In this respect, while methods to tackle this problem are not available, one should be aware of this potential problem for their analyses, interpretation of results and overall conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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
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https://f1000research.com/articles/9-200
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https://f1000research.com/articles/8-1750/v1
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14 Oct 19
|
{
"type": "Software Tool Article",
"title": "Interactive Clustered Heat Map Builder: An easy web-based tool for creating sophisticated clustered heat maps",
"authors": [
"Michael C. Ryan",
"Mark Stucky",
"Chris Wakefield",
"James M. Melott",
"Rehan Akbani",
"John N. Weinstein",
"Bradley M. Broom",
"Michael C. Ryan",
"Mark Stucky",
"Chris Wakefield",
"James M. Melott",
"Rehan Akbani",
"John N. Weinstein"
],
"abstract": "Clustered heat maps are the most frequently used graphics for visualization and interpretation of genome-scale molecular profiling data in biology. Construction of a heat map generally requires the assistance of a biostatistician or bioinformatics analyst capable of working in R or a similar programming language to transform the study data, perform hierarchical clustering, and generate the heat map. Our web-based Interactive Heat Map Builder can be used by investigators with no bioinformatics experience to generate high-caliber, publication quality maps. Preparation of the data and construction of a heat map is rarely a simple linear process. Our tool allows a user to move back and forth iteratively through the various stages of map generation to try different options and approaches. Finally, the heat map the builder creates is available in several forms, including an interactive Next-Generation Clustered Heat Map that can be explored dynamically to investigate the results more fully.",
"keywords": [
"Bioinformatics",
"Genomics",
"Heat Map",
"Web Tool",
"Website",
"Hierarchical Clustering"
],
"content": "Introduction\n\nMany thousands of publications on genomics studies include clustered heat maps (CHMs) because the hierarchical clustering and intuitive visualization provide insight into the relationships among sample sub-groups and key biological processes1–8. Construction of a CHM requires data transformation, application of clustering methods, association of covariate (classification) data, and production of the heat map visualization. Generally, those tasks require the assistance of an analyst with biostatistics or bioinformatics skills who can work in R or a similar language to manipulate the study data and generate the map. This is usually not a simple linear process because data transformation and clustering methods are often revisited to find the ideal match for the study, and modifications are often made to heat map visualizations to select the best colors, adjust covariates, insert gaps, etc. Our Iterative CHM Builder is a web-based tool for generation of high-quality heat maps that can be used by investigators with no bioinformatics experience and only modest exposure to biostatistical methods. The tool guides users through the steps of creating a heat map and supports iterative refinement of the map by working backward and forward through the steps to refine data transformation, annotation, clustering, and formatting options. (Caveat: Iterative exploration of different options may introduce a multiple-comparisons issue that would have to be taken into account if the map were used for formal statistical inference, rather than discovery.)\n\nOne obvious limitation of traditional heat maps is that they contain a huge amount of information but are static in nature and do not readily support a deeper exploration of the biology behind the image. The Iterative CHM Builder produces traditional heat map images as PDF files but can also produce interactive next-generation CHMs (NG-CHMs). NG-CHMs support interactive exploration of patterns in the data through zooming, panning, searching, and advanced link-outs to dozens of external resources. An NG-CHM file can be downloaded and viewed locally with the NG-CHM viewer and, importantly, can be embedded in a study results webpage or publication.\n\nThe iterative CHM Builder9, available at https://build.ngchm.net/NGCHM-web-builder/, is easy to try out using sample data provided at the site. Other methods of producing NG-CHMs, including an R library and a set of tools for the Galaxy platform10,11, are described at https://www.ngchm.net/.\n\n\nMethods\n\nThe Interactive Builder9 is web-based application that accepts an uploaded data matrix and then walks the user through several steps to transform the data, perform hieratical cluster, and format the resulting CHM. The application is implemented as HTML, CSS, and JavaScript on the browser-side and Java servlets on the web server. Data manipulation and heat map generation are implemented in Java classes used by the servlets. The clustering is performed by a servlet using the Renjin engine (https://www.renjin.org) to perform R clustering functions in Java. Browser sessions are tracked by the server to create a working area for each user and prevent users from seeing each other’s data or maps. In addition to the working version of the data matrix on which transformations are performed, an original version of the matrix is preserved. Returning to a previous matrix state is accomplished by restoring the original version and then re-applying transformations until the requested state is restored. The site retains constructed heat maps and the related uploaded data only for the duration of the HTTP session.\n\nA Java NG-CHM heat map generator .jar file is used to construct the heat map repeatedly as options are selected in each step of the builder. The heatmapProperties.json file, which contains all options selected by the user, conveys the selected options to the generator. The current NG-CHM file set is stored in a directory under the session ID. The NG-CHM file is a zipped version of the NG-CHM directory. The downloaded .ngchm file can be saved locally and viewed interactively using a local instance of the NG-CHM viewer that can also be downloaded from the builder site. An overview is given in Figure 1.\n\nHeat maps are built on a webserver. A browser session ID is used to create a separate, temporary working area for each user. Heat map construction sessions are cleaned up when the session is ended, but PDF and NG-CHM heat map files can be downloaded.\n\nThe full source code for the Interactive Builder is available in GitHub.\n\nThere is no need to install software to use the Interactive Builder9 it is available for public use on our server at https://build.ngchm.net/NGCHM-web-builder/. If, however, a local private installation of Interactive Builder is preferred, there are two simple installation methods.\n\nOrganizations familiar with Docker can run the Builder as a Docker container. To do this, clone the git repository. The base folder of this repository has a docker build file. Run the docker build command in this directory with a –t option to name the resulting docker image. Then use the docker run command to start a container using the image. The heat maps created by the software are transient and last only for the duration of a user http session so there is no need to mount an external directory to the container for persistent storage. The port for connecting to the webserver in the container does need to be specified in the docker run command. Connect the desired external port to the tomcat instance in the container, for example –p 80:8080. Users should then be able to connect to Interactive Builder using their browser and the URL of the docker container. For example, http://<docker machine IP or URL>/ NGCHM-web-builder.\n\nThe other option for deploying the software is to install it on an existing web server like tomcat. To do this, first clone the git repository and then use the ant script, ant_buildfile.xml in the NG-CHM_GUI_BUILDER folder to create a .war file. Then simply copy the .war file to the webapps directory of the web server. The application should then be available at http://<server URL>/ NGCHM-web-builder.\n\n\nUse case\n\nThe starting point for a CHM is a matrix of data. In this use-case example, we focus on gene expression data from The Cancer Genome Atlas (TCGA) bladder cancer project12,13. The rows and columns of the matrix require identifiers, in this case sample ids and gene symbols, and the cells of the matrix must be numeric values. The builder will accept either a tab-delimited text file (*.txt), comma-separated text file (*.csv) or Excel spreadsheet (*.xlsx).\n\nThe Open Matrix File button on the first page of the builder (Figure 2) is used to upload the data matrix. A name and optional description to be associated with the heat map are entered. When the data have been loaded, the Select Matrix page will show the first few rows and columns of the matrix. It is important that the builder correctly identify the row labels, column labels, and matrix data; the backgrounds of labels and matrix data should be blue and green, respectively. If the input file has extra rows or columns, you may need to correct the identification of labels and matrix data by selecting the appropriate radio button and then clicking on the correct location in the matrix displayed.\n\nNote that several screens in the builder include advanced features that are hidden by default to simplify the process for first-time users. The use-case example here does not require advanced features, but be aware that additional capabilities can be accessed using the Advanced Features checkbox.\n\nCreating a good heat map depends on proper data preparation. The second step in the build process is the Data Transform page (Figure 3), which provides three primary categories of matrix transformations: functions that identify and replace missing/invalid values, filters to remove rows or columns, and transforms to perform mathematical operations on data values. There are additional choices in advanced mode for transposing the matrix and calculating correlations.\n\nThe right-hand panel of the Transform page provides summary statistics about the data matrix, including the number of rows and columns, a histogram of the data distribution, and an indication of the number of invalid cells in the matrix. The top of the page also provides suggestions about transformations that can be performed and flags any problems with the data. The use-case matrix is too large for the Iterative Builder to use in creating a heat map, so a message in red indicates that. Currently, the website limits the heat map to no more than 4,000 total rows and columns and no more than 3,500 elements on either axis.\n\nFor this use case, we apply the following transforms:\n\nApply a threshold to remove (set to NA) values that are less than 0.00001.\n\nLog Transform Base 10.\n\nMean Center the Rows.\n\nFilter to remove rows with >50% missing values\n\nFilter to keep only the 500 rows with the highest standard deviation. That is done to find the rows that differ the most across samples. Those rows will drive clustering and patterns in the heat map.\n\nAfter applying the transformations, the matrix contains no errors and should be suitable for heat map generation (Figure 4). Note that the left-hand panel shows the history of transformations performed on the matrix, and one can ‘undo’ back to any previous state of the matrix (including the original version) by clicking the desired previous state and hitting reset. More generally, the entire process of creating a heat map is iterative; the Next and Previous buttons can be used to return to previous steps to try different options. If, after generating the heat map, it appears that there should be more or fewer rows or different transforms, one can return to the pertinent screen and use the history and Reset option to adjust the data matrix. Finally, as an added feature, the Transform screen enables the user to download the filtered, transformed matrix for use in other analyses.\n\nThe history of transformations in the left-hand panel can be used to undo changes and revert to previous matrix states.\n\nThe next step is clustering (Figure 5). The row order and column order drop-down menus can be used to select the clustering algorithm and distance measure to be applied to the rows and/or columns. Ward’s algorithm with Euclidean distance metric is one common choice, but the menus include many other possibilities, appropriate for different purposes and data characteristics. For the sample case, the Ward/Euclidean options provide strong separation in the dendrogram and interesting groups of samples. The menus also allow the rows and columns to be left in original order or randomized. Additional options will be provided in the future.\n\nThe Apply button performs clustering and displays the resulting dendrograms.\n\nPlease be aware that clustering of larger matrices may take a few minutes to complete. (The time it takes to cluster data increases approximately as the square of the number of rows or number of columns, whichever is larger.)\n\nThe next page allows covariate (classification) bars to be added to the heat map (Figure 6). Covariate bars add descriptive information about the rows or columns of the heat map. A covariate bar file has the same labels as the rows or columns in the matrix and an annotation value. In this use-case we will use TCGA clinical data to add age, smoking status, gender, and tumor stage to the heat map. The covariate file contains sample ids and clinical values – one value per line. When a covariate file is added, one must identify it as a row or column covariate and specify whether it contains discrete (categorical) data or continuous values. In this case smoker status, gender, and stage are discrete column covariates, and age is a continuous column covariate.\n\nThis screen is also used to change the color of values and ordering of the covariate bars.\n\nAfter covariate bars have been added, the colors associated with the covariate values can be changed. If the color scheme might be useful for other maps, the palette can be saved to the server using the See Palettes button. Covariates can be reordered on the same screen.\n\nAn advanced feature, accessed on the cluster page, is the ability to generate a covariate bar based on the clustering dendrogram. If, for example there are four distinct clusters in the data and one wants to emphasize them in discussion of the heat map, a covariate that identifies the four top clusters based on the four top branches of the dendrogram can be generated.\n\nAnother notable advanced feature is the ability to include classification data in the original matrix uploaded in the first step, rather than providing individual covariate files on the covariate page. Choosing advanced features on the first page enables the user to identify covariates as well as labels and data in the uploaded matrix.\n\nThe format screen (Figure 7) supports the final step in generation of a heat map, adjustments of its appearance:\n\nMany appearance change options are available.\n\nAdjustment of colors and break points in the body of the heat map.\n\nFormatting of labels\n\nFormatting of the dendrograms\n\nSpecification of the data type of the labels for link-outs.\n\nFor this use case, several changes were made: (i) a slight adjustment to the break points to emphasize high and low values in the matrix, (ii) identification of row labels as gene symbols, and (iii) identification of column labels as TCGA sample identifiers. Associating the labels with known data types activates available type-specific link-outs to external data resources.\n\nInteresting advanced features on the same page include the addition of ‘top items’ that will be displayed in the global (i.e., full) heat map view. For example, to show the positions of a few key genes, they can be entered on the page and will show on the global heat map display. Another powerful advanced feature is the ability to add gaps to emphasize sub-groups in the heat map.\n\nThe heat map is now complete, but the Prev button can still be used to go back to previous build steps to try different options. On this final page of the Interactive Builder (Figure 8), the map can be explored dynamically and downloaded. The Get Heat Map PDF button downloads a PDF of the summary and/or detail views as they appear on the screen – including a version of the detailed view zoomed as desired. The legends and other metadata are shown on a separate page of the pdf. The final screen can also be used to explore the dynamic heat map by zooming, panning, searching, dendrogram selection, and link outs. Clicking the Expand Map button devotes the whole browser window to the map.\n\nHeat maps constructed on the Iterative Builder website are not saved. However, NG-CHMs can be downloaded to save and explore dynamically on your own computer. Select the Get NG-CHM file to obtain a map and then select the Get Heat Map Viewer to get a stand-alone NG-CHM viewer to run on your computer. See our NG-CHM site for more details on the capabilities of dynamic heat maps, additional builders to generate NG-CHMs (Galaxy and R)2, and instructions on how to embed dynamic heat maps in your websites - https://www.ngchm.net/. Also see our YouTube channel for tutorials on NG-CHM features.\n\n\n\n\nThe interactive NG-CHM produced by the Builder for the use case can be viewed here. Try the pan, zoom, search, and link-out features.\n\n\nReproducibility\n\nReproducibility of results is becoming increasingly important for publication in high-impact journals14. Therefore, it is important to be able to report the exact steps performed to transform data and create a heat map. That is particularly challenging with an iterative tool that facilitates exploration of alternative options. The Get Creation Log button on the file page of the Iterative Builder is meant to address that need. The history provided by the log shows each option, including the data transformations that were performed to produce the current map. With the original data file and the history, it is possible to recreate a heat map exactly.\n\n\nConclusions\n\nThe Interactive CHM Builder9 is an easy to use yet powerful tool for creating custom clustered heat maps for any type of study that has a matrix of data. It requires no programing skills and has an intuitive step by step process to prepare the data and build high-quality CHMs. Sample data is built-in so it takes just seconds to try out the process and become familiar with the basic steps for heat map generation. It is also easy to back up to previous steps or data states to try alternative approaches and refine formatting. Finally, heat maps may be download as either PDF files or as NG-CHM files that support in-depth exploration of the maps.\n\n\nData availability\n\nOpen Science Framework: NG-CHM Interactive Builder Use-Case Data. https://doi.org/10.17605/OSF.IO/ABJD713.\n\nThis project contains the sample TCGA bladder cancer matrix used in the use-case.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nThe Iterative CHM Builder is freely available for use as a web resource at: https://build.ngchm.net/NGCHM-web-builder/.\n\nSource code available from: https://github.com/MD-Anderson-Bioinformatics/NG-CHM_GUI_BUILDER.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.34606739.\n\nLicense: GNU General Public License version 2.",
"appendix": "References\n\nWeinstein JN, Myers T, Buolamwini J, et al.: Predictive statistics and artificial intelligence in the U.S. National Cancer Institute's Drug Discovery Program for Cancer and AIDS. Stem Cells. 1994; 12(1): 13–22. PubMed Abstract | Publisher Full Text\n\nWeinstein JN, Myers TG, O'Connor PM, et al.: An information-intensive approach to the molecular pharmacology of cancer. Science. 1997; 275(5298): 343–9. PubMed Abstract | Publisher Full Text\n\nMyers TG, Anderson NL, Waltham M, et al.: A protein expression database for the molecular pharmacology of cancer. Electrophoresis. 1997; 18(3–4): 647–53. PubMed Abstract | Publisher Full Text\n\nEisen MB, Spellman PT, Brown PO, et al.: Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 1998; 95(25): 14863–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScherf U, Ross DT, Waltham M, et al.: A gene expression database for the molecular pharmacology of cancer. Nat Genet. 2000; 24(3): 236–44. PubMed Abstract | Publisher Full Text\n\nRoss DT, Scherf U, Eisen MB, et al.: Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet. 2000; 24(3): 227–35. PubMed Abstract | Publisher Full Text\n\nZeeberg BR, Qin H, Narasimhan S, et al.: High-Throughput GoMiner, an 'industrial-strength' integrative gene ontology tool for interpretation of multiple-microarray experiments, with application to studies of Common Variable Immune Deficiency (CVID). BMC Bioinformatics. 2005; 6: 168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeinstein JN: Biochemistry. A postgenomic visual icon. Science. 2008; 319(5871): 1772–3. PubMed Abstract | Publisher Full Text\n\nmstucky, flikeda, Michael Ryan, et al.: MD-Anderson-Bioinformatics/NG-CHM_GUI_BUILDER 2.15.1. (Version 2.15.1). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3460673\n\nBroom BM, Ryan MC, Brown RE, et al.: A Galaxy implementation of next-generation clustered heatmaps for interactive exploration of molecular profiling data. Cancer Res. 2018; 77(21): e23–e26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobertson AG, Kim J, Al-Ahmadie H, et al.: Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. Cell. 2017; 171(3): 540–556.e25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRyan M: NG-CHM Interactive Builder Use-Case Data.2019. http://www.doi.org/10.17605/OSF.IO/ABJD7\n\nMcNutt M: Reproducibility. Science. 2014; 343(6168): 229. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "55133",
"date": "29 Oct 2019",
"name": "Natasha Caplen",
"expertise": [
"Reviewer Expertise Functional genomics. We have relevant expertise in the generation and interpretation of complex ‘Omic scale datasets",
"but not in the statistical analysis that underlays the tool described. Our viewpoint represents that of the potential user of the tool described in this study."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRyan and co-workers have developed the software tool Interactive Clustered Heat Map (CHM) builder to enable investigators with minimal expertise in bioinformatics and biostatistics to generate publication-quality heatmaps. The use of heatmaps to visualize related datasets is a common feature in many reports of the results of studies that include genome or transcriptome-scale experiments. However, the statistical underpinnings of a heatmap require the application of appropriate transformation and clustering procedures. The interactive CHM tool makes use of user-uploaded data that is then processed to generate heatmaps defined by a set of standardized options; for example, the user can select different distance metrics (e.g., the calculation of Euclidean distance versus Manhattan distance) or clustering options (random versus hierarchical). The user also has the option to input possible co-variant data sets for the further stratification of the primary results. Furthermore, the user can customize the visual properties of the heatmap by selecting the output of the computational pipeline from a palette of colors. The article itself is well-written, though, as stated below, we recommend some edits to the current text. A particularly positive feature of this CHM tool is the inclusion of a dynamic capability that allows the user to explore their data in greater depth. Many of the features of the graphical user interface (GUI) are easy to use, and the user does not have to refer to the accompanying article describing the builder software continually. However, to enhance the impact of this resource, we recommend modification of the current versions of their article and software tool to address the following points.\n\nArticle\n\nIn the Introduction, the authors discuss the user’s ability to use their tool to generate heatmaps reiteratively, refining data transformation, annotation, clustering, and formatting. The authors also point out that this may introduce the risk of generating a multiple-comparison issue. To help the user avoid such issues, can the authors briefly mention other resources (e.g., review articles) that the user can refer to when considering which of the transformation, clustering, and distance metrics will be most applicable to their dataset?\n\nThe authors should include a discussion of how the interactive HCM builder compares to other free heatmap generators available, for example, heatmapper.ca; Babickiet al., Heatmapper: web-enabled heat mapping for all Nucleic Acids Res. 2016 May 17 (epub ahead of print). DOI:10.1093/nar/gkw419); and the Morpheus software from the Broad Institute (https://software.broadinstitute.org/morpheus/).\n\nSome datasets require non-hierarchical clustering to obtain the most appropriate and meaningful interpretation of the results. Please explain why this software provides only either hierarchical, random, or no clustering options?\n\nWebsite\n\nSome test runs found that when the user runs through the work-flow and generates a heatmap using a dataset, the generation of a new heatmap either using the same dataset or a different dataset requires the user to close the website and re-open the homepage. The re-set function may need modification.\n\nSome test runs found that when choosing the formatting and then palettes after adding co-variants, the apply button on the left-hand window has lines running through it.\n\nIt is easy to maneuver and resize the highlighter box over any region of the heatmap generated using the sample data. However, we noted not all heatmaps performed as well using user-uploaded data.\n\nPlease state clearly on the website’s front-page that the website limits the heat map to “no more than 4,000 total rows and columns and no more than 3,500 elements on either axis.” In the absence of this statement on the front-page, users may attempt to upload more complex datasets.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5302",
"date": "19 Mar 2020",
"name": "Michael Ryan",
"role": "Author Response",
"response": "Thank you for your detailed comments and suggestions on the article and the tool. Each suggestion/comment is addressed below:Article In the Introduction, the authors discuss the user’s ability to use their tool to generate heatmaps reiteratively, refining data transformation, annotation, clustering, and formatting. The authors also point out that this may introduce the risk of generating a multiple-comparison issue. To help the user avoid such issues, can the authors briefly mention other resources (e.g., review articles) that the user can refer to when considering which of the transformation, clustering, and distance metrics will be most applicable to their dataset? We are not aware of any review article that covers the topic comprehensively. But, in the text, we do cite an article of ours (Weinstein JN: A postgenomic visual icon. Science. 2008;319: 1772) that provides additional background on some of the relevant subtleties of heat map generation. As you correctly point out, the optimum approach depends on the specifics of an individual dataset and objectives of the study. In light of your comment, we will consider writing a review article that addresses those issues at more length. Also, we are contemplating a future enhancement to the Interactive CHM Builder that would provide templates or workflows based on study type as a starting point to assist with navigating data transformations and heat map generation.The authors should include a discussion of how the interactive HCM builder compares to other free heatmap generators available, for example, heatmapper.ca; Babickiet al., Heatmapper: web-enabled heat mapping for all Nucleic Acids Res. 2016 May 17 (epub ahead of print). DOI:10.1093/nar/gkw419); and the Morpheus software from the Broad Institute (https://software.broadinstitute.org/morpheus/). Thank you for the suggestion. We have added a paragraph to the conclusions section to enumerate what we feel are the advantages of our Interactive CHM builder compared with the other similar tools.Some datasets require non-hierarchical clustering to obtain the most appropriate and meaningful interpretation of the results. Please explain why this software provides only either hierarchical, random, or no clustering options?We agree. Thank you. The methods we have currently implemented are the ones that are most heavily used in publications of omics research. We will add non-hierarchical clustering methods to our requested features list for a future release.Website Some test runs found that when the user runs through the work-flow and generates a heatmap using a dataset, the generation of a new heatmap either using the same dataset or a different dataset requires the user to close the website and re-open the homepage. The re-set function may need modification. Thank you for reporting this issue. We have modified the restart flow and believe the problem has been corrected.Some test runs found that when choosing the formatting and then palettes after adding co-variants, the apply button on the left-hand window has lines running through it. We have been unable to reproduce that issue in the latest release of the software so we believe it has been corrected. If you encounter it again, we would appreciate it if you submit a git issue, noting the browser and operating system for which it occurs.It is easy to maneuver and resize the highlighter box over any region of the heatmap generated using the sample data. However, we noted not all heatmaps performed as well using user-uploaded data.Thank you for the report. Since submission of the paper, we have made several improvements to the selection/sizing features and have tested many odd sized asymmetrical matrices. We will continue to implement improvements in selection mechanics if additional issues arise. Please state clearly on the website’s front-page that the website limits the heat map to “no more than 4,000 total rows and columns and no more than 3,500 elements on either axis.” In the absence of this statement on the front-page, users may attempt to upload more complex datasets.For many studies, an important step in preparing data for clustering and heat map generation is filtering out rows and/or columns that have a high proportion of missing values or that show little variance across samples. We want to allow users to upload matrices that are above the clustering limit because the filtering step will often reduce the size of the matrices such that they can be clustered. The manuscript was not clear on this point. Thank you for pointing this out. We have modified the Use Case section “Transform/filter the data”, paragraph 2, to explicitly discuss clustering limits and the use of filtering to reduce larger datasets.The interactive nature of the tool does limit the maximum matrix we can cluster. As you know, the compute time for most clustering algorithms essentially increases as the square of the largest dimension. The tool’s limit for the clustering step has been increased from 4,000 to 5,000 total rows/columns. The 3,500 axis limit has been removed. We’ve also added new system messages that more clearly explain those issues, and we plan to continue pursuing increases in the limits as computational power increases and clustering algorithms advance."
}
]
},
{
"id": "59179",
"date": "21 Feb 2020",
"name": "Melissa Cline",
"expertise": [
"Reviewer Expertise 20 years of experience in genomics",
"including RNA expression analysis and cancer data visualization."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRyan et al. present a manuscript for the interactive clustered heat map builder tool that has been widely used in cancer research consortia. The tool is of excellent quality overall, is very useful, and is intuitive in its design and execution. The manuscript is well-written, with some caveats below.\nMajor feedback: The authors are not doing justice to the tool, which offers much more than user-friendly heatmap generation. To put the functionality in perspective, they should contrast it with the Cluster/TreeView suite, which also offers a user-friendly interface to filtering and data transformation.\nMinor feedback\nThe Operation subsection assumes knowledge of Docker and Tomcat. The authors should cite appropriate background reference material for readers who aren't familiar with these technologies.\nFor the use case, the authors summarized how they transformed the data, but did not indicate how those transformations were done with their tool. This needs to be clarified, because it's not obvious.\nThe sample data in the OSF Storage site is stored as a single tarball. This is awkward, as the entire tarball has to be downloaded and expanded in order to access any single file.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5303",
"date": "19 Mar 2020",
"name": "Michael Ryan",
"role": "Author Response",
"response": "Thank you for your feedback and suggestions. Below we have described how each was addressed.Major feedback:The authors are not doing justice to the tool, which offers much more than user-friendly heatmap generation. To put the functionality in perspective, they should contrast it with the Cluster/TreeView suite, which also offers a user-friendly interface to filtering and data transformation.Good suggestion. We have modified the 5th sentence in the first paragraph of the introduction to broaden the description of the scope of the tool and have included a new paragraph in the conclusion section to contrast our tool with Cluster 3.0/Treeview.Minor feedbackThe Operation subsection assumes knowledge of Docker and Tomcat. The authors should cite appropriate background reference material for readers who aren't familiar with these technologies.We agree. Additional detail including links to the appropriate reference material for Docker and Tomcat, has been added to the Operation section.For the use case, the authors summarized how they transformed the data, but did not indicate how those transformations were done with their tool. This needs to be clarified, because it's not obvious.Thank you for pointing that out. The transforms section of the use case has been modified to provide the exact path through the screen options for each transform performed. That should make it easier to follow the steps exactly.The sample data in the OSF Storage site is stored as a single tarball. This is awkward, as the entire tarball has to be downloaded and expanded in order to access any single file.Agreed. Accordingly, we have modified the OSF Storage site to contain a folder with all of the files needed for the use case as individual, uncompressed files."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1750
|
https://f1000research.com/articles/9-197/v1
|
19 Mar 20
|
{
"type": "Research Article",
"title": "Trends and determinants of contraceptive method choice among women aged 15-24 years in Kenya",
"authors": [
"Wambui Kungu",
"Alfred Agwanda",
"Anne Khasakhala",
"Alfred Agwanda",
"Anne Khasakhala"
],
"abstract": "Background: Studies show a gap in addressing the reproductive health goals of younger women whose inconsistent use of contraception is high in spite of their great need for it. The women aged 15-24 present high potential for unintended pregnancy and increase the challenge for retention of users which is key in maintaining and pushing up the current gains in contraceptive prevalence rate (CPR). Objective: The objective of the study was to examine trends in contraceptive method choice for young women aged 15-24 years using modern methods and to determine factors associated with their choices. Methods: The study used data from KDHS of 2003, 2008/9 and 2014 in descriptive analysis and logistic regression to determine the socio-economic variables that influence the choice of contraceptive methods for young women. Results: Results showed a general shift in use towards long term modern contraceptives with the shift being more pronounced among young women with primary education, from rural areas, lower wealth households, and low contraceptive use regions. Women with secondary education and higher wealth status are shifting towards short term methods. Findings confirmed socio-demographic factors of age, education, wealth status and type of region as predictors of contraceptive use. Conclusions: Contraceptive information and services should be enhanced for young women to make informed choices concerning their reproductive and sexual health to enable them complete school and transition to colleges to acquire relevant skills that will make them optimally productive and lead Kenya to achieving the demographic dividend.",
"keywords": [
"Trends",
"Determinants",
"Contraception Method Choice",
"15-24",
"Kenya"
],
"content": "Introduction\n\nKenya has invested considerable resources towards improving adolescent sexual reproductive health (ASRH) and has in place the 2015 National Adolescent Sexual Reproductive Health (NASRH) Policy; however, the rates of teenage pregnancies do not reflect these heavy investments and are a matter of public health and social concern. Teenage pregnancy rates have plateaued for over a decade and have persisted at 20% between 2003 and 20141–3.\n\nMore than 10,000 Kenyan girls quit school every year due to unintended pregnancies and more than 10 percent of births in Kenya occur to girls aged 15–19 years4. Unintended pregnancies among teenagers are linked to poor health outcomes, which include sexually transmitted infections, unsafe abortion, miscarriages, stillbirths and complications during births that can lead to infant and/or maternal deaths. There is also rapid-repeat pregnancy, which is linked to greater risks of morbidity and mortality in mothers and new-borns as well as poor mental health5.\n\nThe Kenya Demographic and Health Survey (KDHS) 2014 revealed critical facts about adolescents in Kenya. One out of every three (30%) adolescent girls reported having had sex while the average age at which adolescent girls had their first child was 17 years and 12% of the girls were already in a relationship. Most sexually active 15–19-year-olds do not use contraception and only about 50% of those on contraception use it consistently with 14% of them being ambivalent about pregnancy. As many as 90% of unmarried sexually active adolescent girls reported not wanting to get pregnant in the next two years but only 43 percent were using a method to prevent pregnancy. Average unmet need for family planning for the group was 21% against the national average of 18%1,6. Several interventions to address ASRH needs have been put in place but the median age at first birth continues to decrease.\n\nKenya has a very youthful population with the 15–24-year-old age cohort constituting about 30% of the population of 47 million7. The 15–24-year-old young women take about 15% which comes to 7 million. With the economic growth far below the desired 10% per annum, this group is critical to economic development but for them to be substantially productive the problems surrounding them have to be comprehensively addressed8.\n\nDemographic dividend has been proposed as a viable solution to economic challenges faced by developing countries. Kenya’s demographic window is expected to open in 2038 and it could achieve demographic dividend by 2050 if it invests in the huge youth population and especially the adolescents. Demographic dividend modelling shows Kenya can attain $4,595 in GDP per capita by 2050 if it makes strategic investments against $896 if it maintains the status quo. This would mean a staggering fivefold economic growth whose achievement, family planning can accelerate9,10.\n\nAlso, improved access to reproductive health has been identified as critical in the process of development in Sustainable Development Goal (SDG) 3.711. Adolescent and young women childbearing is linked with lower educational achievement and could lead to a cycle of poverty and hinder achievement of demographic dividend. Meeting the contraception needs of adolescent and youth to prevent unintended pregnancies should thus be a priority12.\n\nThe Health Act of 2017 and constitution of Kenya have enshrined the right of every woman to safe, affordable, effective, and acceptable contraception services13. FP2020 has issued the Global Consensus Statement to expand contraceptive options for adolescents and youth and set higher family planning targets14. Family planning documents in Kenya advocate for promotion of long acting reversible contraception (implants and IUD) as safe, convenient and highly effective methods.\n\nKDHS data shows the 15–24 age group taking a sizeable share of the modern method mix in Kenya among all users of reproductive age with 15% pill users, 8% of IUD use, 18% implants use and 24% injection use1. It also shows shifting popularity from injections to implants for the young women, but correct and consistent use remains a challenge and unintended pregnancies prevail5. Data is constantly needed to monitor the success of the many policy and program efforts towards effective reproductive health programs for youth.\n\nPoverty is a major contributing factor to teenage pregnancies with girls from poor households more exposed to the risk of early childbearing8. Evidence has shown that without contraception, more than 90% sexually active young women get pregnant within a year15. The leading reason for adolescents’ delay or for not obtaining contraceptive services is apprehension on confidentiality and side effects.\n\nIn Kenya, 22% of unintended pregnancies end up in dangerous abortions for adolescents because they mostly abort during the second trimester after late discoveries of the pregnancies. Adolescents below 19 years make up 20% of women seeking post-abortion care services in health facilities and 50% admissions from severe complications16,17. There is urgent need to expand access to safe and affordable reproductive health care and options especially for the more vulnerable young women of lower education and economic status who procure unsafe abortions more18.\n\nEvidence has shown increased use of contraception is a primary factor in reduction of adolescent fertility and births rates19. Comprehensive sexuality education (CSE) and integrated youth friendly services have also shown success when well implemented but youth centres and peer education have not been effective20. Providing contraceptive information, counselling and services and the use Larcs has been found useful to encourage new acceptors of contraception among nulliparous young women. LARCs are 99% effective if correctly used and have several non-contraceptive benefits like reducing menstrual flow and pain and thus endometriosis.\n\nTo achieve demographic dividend, a country must undergo a demographic transition which brings about lower birth and death rates through investments in family planning. Significant declines in fertility can be reaped from a high contraceptive use momentum that young women can generate. A slower population growth can also be achieved if age at first birth is delayed for most of these women. A huge young population represents great economic potential and investments in their health and education can stimulate new economic opportunities21.\n\nPrevious studies in Kenya have identified socio economic factors such as education and wealth as significant on the use of contraception22,23. Choice and continued use of a method is influenced by socio-economic factors hence the choice of explanatory variables24. This study attempted to provide a current information base on how contraceptive use among 15–24 years old girls in Kenya has evolved from 2003 to 2014 with a view to providing evidence which could form a basis to increase modern contraceptive use among them.\n\nThe main objective of the study was to examine factors that influence the choice of contraceptive methods for young women 15 to 24 years old.\n\n\nMethods\n\nThis study analysed secondary data from the Kenya Demographic and Health Surveys of 2003, 2008/9 and 2014, which contained detailed contraceptive calendars for all interviewed women aged 15 to 49 years. The study was based on the samples of all 15-to-24-year-old women, whether married or single, who reported current use of modern contraception within the five years prior to the three surveys. It excluded those who were not using a main modern contraceptive at the time of the each KDHS. Data was specifically obtained from the variables on current method used, age, education, residence, wealth and region.\n\nData analysis was performed using SPSS version 22. The study started with obtaining frequencies for each of the methods to obtain a trend analysis of the main methods of contraception used by the young women, namely the Pill, IUD, Implants, Injection and Condoms for the three data sets. Afterwards, young women contraceptive users were profiled using cross-classification analysis on socio demographic variables of age, education, wealth status, place of residence and type of region to provide trends of contraceptive method choice within the period of study. This refers to arranging the data into classes according to the variables. Pearson’s Chi square test was used to determine existence of any significant relationships between the dependent variable (contraceptive method choice) and the independent socio demographic variables.\n\nThe dependent variable was dichotomous, hence binary logistic regression was used to determine the net effects of the independent variables (age, education, wealth status and type and region of residence) on method choice.\n\nContraceptive method was recoded as either short term (pills, condoms) or long term (injection, IUD, implants), while level of education was recoded into two categories (primary and secondary/higher). Household wealth status was recoded into three tertiles of lower (lowest, and second lowest), middle (middle) and higher (fourth quintile and highest) while region was recoded into two categories of high contraceptive use (Nairobi, Central and Eastern) and lower contraceptive use (the rest of the regions).\n\n\nResults\n\nThe first objective of the study was ‘to establish the profiles of 15–24-year-old modern contraceptive users’ over the years 2003 to 2014. The following Table 1 presents the background characteristics of the study population who reported using a main modern method in each KDHS.\n\nThe majority of the study group in 2003 were in the 20–24 age group (76%); this proportion increases to over 80% in 2008/09 and 2014. There is also a trend of rising education levels: in 2003, 68% had received only primary education, which reduces to 51% by 2014; those with secondary education increased from 32% in 2003 to 49% in 2014. The majority of surveyed individuals (63%) were living in rural areas between 2003 and 2008/09, but this figure drops to 51% in 2014. Urban areas accommodated 37% in 2003 and 2008/09 and 49% in 2014.\n\nOn wealth status in 2003 and 2008/09, 20% were in the lower category, 18% in middle and 60% in higher. In 2014, the higher category reduced to 49%, middle increased to 21% and lower to 30%. The emerging trend is increasing users of contraception are of lower wealth status. For region, those from regions with low contraception uptake were the majority at 55–58%, while regions of high contraceptive use made up 42–45% of the surveyed. Regarding the contraceptive methods used, the majority (60%) reported using long-term methods in 2003 and 2008/09, a figure that increased to 76% in 2014; use of short-term methods dropped from 40% (2003) to 27% (2014).\n\nThe general profile of modern contraceptive users among the group is that of women with primary education, living in urban areas, from higher wealth households and regions of low contraception.\n\nThe section addresses the second objective, ‘to establish the trends in contraceptive method choice for young women’, 15–24 in Kenya from 2003 to 2014. Further analysis established shares of use for the Pill, Condom, Injection, IUD and Implants presented in the following figure.\n\nAs seen in Figure 1, the main method of contraception was injection with a share of 54–58% in the three data sets. Its share increased slightly between 2003 and 2008/09 from 56% to 58% then reduced to 54% in 2014. The pill was second most common in 2003 (23%) but declined to fourth (11%) in 2014.\n\nSource: KDHS Data.\n\nCondom use increased from 17% in 2003 to the second largest share in 2008/09 at 24% but fell to third position in 2014, below implants. Implants were fourth between 2003 and 2008/09 with a 2–3% share but between 2008/09 and 2014, had a huge upsurge of 16 percentage points from 1.9% to 17.7% to move to second position after injection. The IUD had a very low share of between 0.4% and 2%.\n\nThe majority of users of long-term contraceptive methods had primary education and lived in rural areas and in regions of lower contraception (Table 2). On wealth status, majority were in the middle category in 2003 but in 2008/09 and 2014, bigger proportions were of lower wealth. Users of short-term methods had secondary education, were wealthy and lived in urban areas mostly in regions of high contraception.\n\nLogistic regression results are presented Table 3. They respond to the third objective of the study, ‘to identify determinants of contraceptive choice among 15–24 year olds.\n\n*<0.05, **< 0.005 ***<0.001. ref, reference category.\n\nResults show that age and education were significantly associated with method choice in 2003. Age was positively associated with choosing a long-term method for a 20–24-year-old woman, being two and a half times more likely than for a 15–19 woman. Education was negatively associated with choosing a long-term method, with women with secondary education being 60% more likely to choose short-term methods than those with primary education.\n\nIn 2008/09, age, education and region were significantly associated with choosing a long-term method, with region and age having positive relationships while education continued to exhibit a negative relationship. For age, there was an almost 100% increase in the odds of a 20–24 woman choosing a long-term method when compared with a 15–19-year-old. Conversely, there was a great decrease in the odds of using a long-term method as the level of education rose. Secondary-educated women were 70% less likely to choose long-term methods relative to those with primary education suggesting an inverse relationship. For region, the odds of a woman from a region of high contraception uptake choosing a long-term method were almost three times greater than for a woman from a region of low contraception uptake.\n\nIn 2014, four variables (age, education, wealth status and region) emerged as significant predictors of contraceptive method choice. Age was positively associated while education, region and wealth status were negatively associated. On age, 20–24-year-old women were almost twice as likely to choose a long-term method when compared with their 15–19 cohorts. Education showed a 60% decrease in the odds of a woman with secondary education choosing a long-term method relative to a woman with primary education. Thus women with secondary education were more likely to choose short-term methods than their less educated counter parts.\n\nRegion had a negative relationship with method choice and emerged as a very significant explanatory variable influencing method choice. There was a 35% decrease in the odds of a woman from a high contraception region choosing a long-term method when compared with a woman from a region with low contraception uptake. Women from regions of high contraceptive use were thus 65% more likely to choose short-term methods than those from regions of lower contraception uptake. Thus, women from high contraception regions were more inclined to use short-term methods.\n\nThose from higher wealth status were less likely to use long-term methods of contraception than those of lower wealth. Results show the odds of choosing a long-term method decreased with increased household wealth. Young women of higher wealth emerged as 40% less likely to choose a long-term method as compared to those from lower wealth status. Women of higher wealth were 60% more likely to choose short-term methods than those of lower wealth.\n\nThere was no notable influence on the choice of a modern method for residence in the regression in all the data sets, suggesting residence could not be used to significantly explain urban-rural variations in contraceptive method choice.\n\nOverall in the regression analysis, age, education, wealth status and type of region emerged as significant predictors of contraceptive method choice.\n\n\nDiscussion\n\nThe study has shown use of modern methods among women aged 15–24 years has been increasing from 2003 but more so from 2008/09. Proportions using modern methods increased almost sixfold between 2008 and 2014. Use of modern methods has been increasing globally and in Kenya in recent years for all women of reproductive age, but more so among those aged 15–24 years, where there are huge numbers of sexually active youth who are initiating contraception. The findings are collaborated by the annual FP2020 reports for Kenya25. Other studies had reported rising use of modern methods and more so injectables earlier5,26.\n\nUse of long-term methods increased by around four times between 2008/09 and 2014. This is not only a Kenyan phenomenon but a general trend in Sub-Saharan Africa, where successful LARC promotion campaigns and innovative youth sexual and reproductive health programs are increasing the numbers of new acceptors. Implants are now the fastest rising method of contraception among women 15–24, their use more than doubling from 10 to 21% between 2008/09 and 2014. Rapid increase in the use of implants has been reported recently for several countries, including Kenya, where scale up campaigns have been conducted27,28. LARCs are more effective and cheaper in the long term than shorter-acting contraceptives, and are recommended for most women, including adolescents and nulliparous women29.\n\nThe shift away from short-term methods resulted in declining use of pills and condoms, as shown in Figure 1 and in latter analysis, as declining use of short-term methods which were previously very popular with young women. Condoms are an important dual contraceptive method for prevention against pregnancy and also sexually transmitted infections and HIV/AIDS. They have been previously marketed aggressively to ensure demand creation but shifting priorities of HIV funding have led to fewer campaigns from 2007, which has probably led to their declining use30.\n\nAs shown in the trend analysis for individual modern methods, implants are the new contraception frontier for women aged 15–24. Other studies have shown implants as increasing their acceptance among young women as efforts to promote them increase. A study in Kenya to promote LARCs among young women showed a major upsurge of implants between 2008 and 2014 with use doubling in the 15–19 age category and increasing by six times among the 20–24-year-old age group31.\n\nRegions of lower contraceptive use are surpassing regions of high contraception and are showing potential for major increases in use of modern contraceptives for women aged 15–24 and it is collaborated by FP2020 progress reports. This presents evidence of potential for scaling up modern CPR and achieving the FP2020 targets25.\n\nRegion was a socio-economic predictor of contraceptive choice in this study in 2008/09 and 2014. However, the classification of regions in terms of contraceptive use, as performed here, has been changing as seen from KDHS 2014 such that regions which previously had lower contraceptive uptake like Western are recording very high uptake of modern contraception especially for LARCs. This is possibly an outcome of high-level campaigns to promote long-term methods in the whole country but more so in the regions of lower contraceptive use.\n\nOn residence, no distinct variations in method choice are seen in rural or urban areas, suggesting good progress towards equity in accessing methods in both types of residence. Previous studies in Kenya had revealed distinct regional differentials showing the likelihood of using a modern method, more so long-term ones, being higher in urban than rural areas and also higher in regions of high contraception uptake than in regions of lower contraception22. In another study in Kenya, residence was also found to influence variations in contraceptive choice5.\n\nThe findings on the significance of education and wealth status are in line with earlier studies in Kenya that found education and wealth had significant influence on the use of contraception22,23. A study in Uganda found similar results24. In contrast, other studies on Kenya have not found education and household wealth to be significantly associated to contraceptive use32,33.\n\nResults have shown that users of modern contraception methods are exhibiting higher education levels in recent years. This is probably a reflection of the heavy investments in education Kenya is making to ensure all children complete primary school and transit to secondary school, and also introducing free primary and secondary education. However, the results of regression with education showed an inverse relationship between education and choice of long-term contraceptive methods in all the data sets. Women with secondary education were 40–70% less likely to choose long-term methods than those with primary education. The results tally with surprising recent findings in Ghana that women with secondary education were using less effective, short-term and periodic contraception more than effective, long-term, modern methods33,34. Questions arise whether they are experimenting with methods, are wary of side effects or are intending to get pregnant.\n\nAn interesting finding is that users of more modern contraception were in the lower wealth brackets in 2008/09 and 2014 and users of long-term methods were more in rural areas. This could imply success of family planning campaigns in rural and low-income areas or improved access for women of lower wealth status. Community health workers (CHWs) whose role in expanding contraceptive access has been revitalized work in some of these rural and hard to reach areas where most households are of lower wealth.\n\nAs observed in the study results, 15–24-year-old users of modern methods are shifting away from short-term contraceptives towards LARCs, which are more efficient, convenient and offer greater user satisfaction35. The shift is more pronounced among women with primary education, from rural areas, lower-wealth households and regions of low contraceptive use. Implants may overtake the injection as the most popular method for this group if the surge in popularity continues.\n\nThe study results generally indicate success for the aggressive Kenyan family planning program and FP2020 campaigns and existence of great potential for scaling up use of long-term methods among the study group. The phenomenal rise in use of long-term methods observed in the review period will most likely continue due to the ongoing global and local promotion towards LARCs and also because of the momentum of demand from the huge population of 15–24 year olds.\n\nSome program and policy implications arise from the study findings. Young women are shifting towards long-term methods and the use of implants. These are provider-dependent methods and therefore the government needs to invest more in ensuring enough skilled providers to handle insertions/removal as well as counselling on any associated side effects. It is expected that implants, because they are long-term methods, will reduce discontinuations and consequently unintended pregnancies among the young women.\n\nThe shift towards long-term methods spells success for the policy on promotion of LARCs for all women, including adolescents. This should increase retention of users of modern methods, protect the gains made in CPR and modern CPR, and contribute towards further reductions in fertility. The cost of LARCs is higher initially, but they are cost-effective in the long term, meaning more funding should be provided towards generating sustainable commodity stocks.\n\nThe changing profile of the users of long-term methods, who mostly live in rural and low-income areas, show potential for increased uptake and stepping up the campaigns for LARCs in these areas with more involvement of CHWs.\n\nThe emerging shift by secondary educated, wealthier women towards short-term methods calls for targeting of the group to minimise unwanted fertility.\n\nThe declining use of condoms raises concern whether the decline will result in increasing incidences of HIV/AIDS and roll back the gains in the fight against HIV/AIDS.\n\n\nConclusions\n\nResults from the study have provided a useful profile of young modern contraceptive users and established a general pattern of their contraceptive use. The study has identified the predictors of contraceptive use among young girls as age, education, household wealth and type of region, and detailed how these factors are changing with time. This information is useful for managers of youth reproductive health programs and could guide interventions to help reduce unintended pregnancies among young girls in Kenya so that girls can stay in school longer and complete their education to desired levels.\n\nThe results for the young women, 15–24 years of age, conform to the general shift towards long-term methods for all women of reproductive age, as seen in the recent studies35,36. Kenya is an example of family planning policy success but the journey towards replacement level fertility cannot be complete if the 15–24 age group is left behind. More investment towards successful adolescent and youth reproductive health are needed to ensure Kenya takes advantage of the opening of the demographic window and reaps the demographic dividend. Young women aged 15–24 are at the centre of these efforts; family planning policy and program managers must ensure they take control of their reproductive health and use contraceptive information and services to their advantage.\n\nContraception allows young women to make informed choices concerning their reproductive and sexual health, stay in school longer, complete higher levels of education and transition to colleges for labour relevant skills that will enable them optimally contribute to the economy and lead Kenya to achieving the much desired demographic dividend. LARC has potential advantages for these women who wish to delay pregnancy for several years.\n\nTo improve the environment for reproductive health and modern contraception for women 15 to 24 years with a view to minimising unintended pregnancies, several interventions can be put in place: 1) Implement fully the National Adolescent Sexual and Reproductive Health Policy, 2015; 2) Initiate age appropriate Life Skills Education (LSE) in all schools; 3) Mainstream youth friendly services into regular services at all health facilities; 4) Expand the range of available method mix for married and unmarried young women and especially the LARCs; and 5) Ensure commodity security by reducing stock outs of commodities.\n\n\nData availability\n\nThe datasets analysed during the current study are available from the MEASURE DHS repository, (http://www.measuredhs.com). Access to the dataset requires registration and is granted to those that wish to use the data for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm.",
"appendix": "References\n\nKenya National Bureau of Statistics: Kenya Demographic and Health Survey 2014. Kenya National Bureau of Statistics and ICF Macro. 2015. Reference Source\n\nKenya National Bureau of Statistics: Kenya Demographic and Health Survey 2008/09. Kenya National Bureau of Statistics and ICF Macro. 2010. Reference Source\n\nKenya National Bureau of Statistics: Kenya Demographic and Health Survey 2003. Kenya National Bureau of Statistics and ICF Macro. 2004. Reference Source\n\nObare F, Birungi H, Chi-Chi U, et al.: Levels, Trends and Determinants of Contraceptive Use among Adolescent Girls in Kenya. Population Council. Aphia II Operations Research Project/Population Council. 2011. Publisher Full Text\n\nNorton M, Chandra-Mouli V, Lane C: Interventions for Preventing Unintended, Rapid Repeat Pregnancy among Adolescents: A Review of the Evidence and Lessons from High-Quality Evaluations. Glob Health Sci Pract. 2017; 5(4): 547–570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrückner H, Martin A, Bearman PS: Ambivalence and pregnancy: adolescents' attitudes, contraceptive use and pregnancy. Perspect Sex Reprod Health. 2004; 36(6): 248–257PubMed Abstract\n\nNCPD: State of Kenya Population Report 2017. National Council for Population and Development. 2018.\n\nNCPD: National Adolescent and Youth Survey. National Council for Population and Development. 2015. Reference Source\n\nNCPD: Kenya’s Demographic Dividend Roadmap. National Council for Population and Development. 2017. Reference Source\n\nHealth Policy Project: Impact Now Model: Estimating the Health and Economic Impacts of Family Planning Use. Health Policy Project (HPP), United States Agency for International Development (USAID) and Marie Stopes International (MSI) and Futures Group. Health Policy Project. 2014. Reference Source\n\nNew J, Cahill N, Stover J, et al.: Levels and trends in contraceptive prevalence, unmet need, and demand for family planning for 29 states and union territories in India: a modelling study using the Family Planning Estimation Tool. Lancet Glob Health. 2017; 5(3): e350–e358. PubMed Abstract | Publisher Full Text\n\nGuttmacher Institute: Adding it Up: Costs and Benefits of Meeting the Contraceptive Needs of Adolescents in Developing Regions. Guttmacher Institute. 2018. Reference Source\n\nGoK: Health Act No. 21 of 2017. Government of Kenya. 2017. Reference Source\n\nFP2020: Global Consensus Statement for Expanding Contraceptive Choice For Adolescent And Youth To Include Long Acting Reversible Contraception. FP2020. 2015. Reference Source\n\nTemple-Smith M, Sanci L: LARCs as first-line contraception - What can general practitioners advise young women? Aust Fam Physician. 2017; 46(10): 710–715. PubMed Abstract\n\nGuttmacher Institute: Adolescents’ Need for and Use of Abortion Services in Developing Countries. Guttmacher Institute. 2016. Reference Source\n\nSedgh G, Finer L, Bankole A, et al.: Adolescent pregnancy, birth, and abortion rates across countries: levels and recent trends. J Adolesc Health. 2015; 56(2): 223–230. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKHRC and RHRA: Teenage Pregnancy and Unsafe Abortion: The Case of Korogocho Slums. Kenya Human Rights Commission and Reproductive Health and Rights Alliance. 2010. Reference Source\n\nLindberg L, Santelli J, Desai S: Understanding the Decline in Adolescent Fertility in the United States, 2007-2012. J Adolesc Health. 2016; 59(5): 577–583. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChandra-Mouli V, Lane C, Wong S: What Does Not Work in Adolescent Sexual and Reproductive Health: A Review of Evidence on Interventions Commonly Accepted as Best Practices. Glob Health Sci Pract. 2015; 3(3): 333–340. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGribble J, Bremner J: The Challenge of attaining the Demographic Dividend. Policy Brief. Population Reference Bureau. 2012. Reference Source\n\nMagadi MA, Curtis SL: Trends and determinants of contraceptive method choice in Kenya. Stud Fam Plann. Population Council. 2003; 34(3): 149–159. PubMed Abstract | Publisher Full Text\n\nKimani M, Njeru M, Ndirangu G: Regional variations in contraceptive use in Kenya: Comparison of Nyanza, Coast and Central Provinces. Afr Popul Stud. 2013; 27(1): 43–52. Publisher Full Text\n\nBbaale E, Mpuga P: Female Education, Contraceptive Use, and Fertility: Evidence from Uganda. Consilience: The Journal of Sustainable Development. Gribble. 2011; 6(1): 20–47. Publisher Full Text\n\nFP2020: Annual Progress Report. Kenya. 2015. Reference Source\n\nHarbison S, Adetunji J: Contraceptive Choice and Discontinuation in Selected African Countries: A Focus on Injectables. 2009. Reference Source\n\nRoss J, Keesbury J, Hardee K: Trends in the contraceptive method mix in low- and middle-income countries: analysis using a new \"average deviation\" measure. Glob Health Sci Pract. 2015; 3(1): 34–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeyonzo N, Nyachae P, Kagwe P, et al.: From Project to Program: Tupange's Experience with Scaling Up Family Planning Interventions in Urban Kenya. Reprod Health Matters. 2015; 23(45): 103–13. PubMed Abstract | Publisher Full Text\n\nTsui AO, Brown W, Qingfeng Li: Contraceptive Practice in Sub-Saharan Africa. Popul Dev Rev. PMC. 2017; 43(7). PubMed Abstract | Publisher Full Text | Free Full Text\n\nMann Global Health: The Condom Program Pathway. Bill and Melinda Gates Foundation. 2017. Reference Source\n\nHubacher D, Olawo A, Manduku C, et al.: Preventing unintended pregnancy among young women in Kenya: prospective cohort study to offer contraceptive implants. Contraception. 2012; 86(5): 511–7. PubMed Abstract | Publisher Full Text\n\nFotso J, Izugbara C, Saliku T, et al.: Unintended pregnancy and subsequent use of modern contraceptive among slum and non-slum women in Nairobi, Kenya. BMC Pregnancy Childbirth. 2014; 14: 224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarson C, Stanfors M: Women’s Education, Empowerment, and Contraceptive Use in sub-Saharan Africa: Findings from Recent Demographic and Health Surveys. Afr Popul Stud. 2014; 28(2): 1022–1034. Publisher Full Text\n\nMarston C, Renedo A, Nyaaba GN, et al.: Improving the Measurement of Fertility Regulation Practices: Findings from Qualitative Research in Ghana. Int Perspect Sex Reprod Health. 2017; 43(3): 111–119. PubMed Abstract | Publisher Full Text\n\nJacobstein R: Liftoff: The Blossoming of Contraceptive Implant Use in Africa. Glob Health Sci Pract. 2018; 6(1): 17–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHP: Increasing Larc uptake in Kenya through Improved Demand Creation Strategies and Provider Support. Best practices from the WHP 2009-2014. Women’s Health Project Best Practices in Kenya. 2015. Reference Source"
}
|
[
{
"id": "61496",
"date": "01 Apr 2020",
"name": "John Cleland",
"expertise": [
"Reviewer Expertise Demography with special interest in fertility and contraception."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents an analysis of method choice among current users of contraception aged 15-24 years, using data from the three most recent KDHSs. Descriptive data on method-specific use are shown but the main analysis makes the binary distinction between long- and short-acting methods. In this age group, the main message is that the share of pills and condoms use in overall modern method protection has declined but the share of implants has increased.\n\nThe main limitation of the paper is the omission of two key influences on method choice in this age band: marital/cohabitation status and motherhood status. The influence of marital status is critical. The 2014 KDHS shows that about 30% of sexually active single women aged 15-24 use condoms compared with only 2% of married women in this age band. Obvious reasons for this difference exist. For young single women sex is often infrequent and clandestine and risks of STI/HIV infection appreciable. Continuous protection from a hormonal method may seem excessive and offers no protection from infection. Young married women are also at risk of STIs but condom use is extremely difficult to sustain within a cohabiting relationship because coital frequency is high and trust is imperative. Without this variable in the analysis, the results are very difficult to interpret. For instance, is the negative association between length of schooling and use of long-acting methods simply a consequence of the fact that well-educated young women are less likely to be married than their less educated counterparts.\n\nThe omission of motherhood status is perhaps less crucial but there are reasons to anticipate an influence on method choice. Considerable ethnographic evidence exists that women (single or married) may be reluctant to use a hormonal method before giving birth because of fears that use may jeopardise future fertility. Mothers may also find access to clinical methods easier than non-mothers.\n\nMy conclusion is that the analysis should be re-done with marital status as a predictor and at least some exploratory analysis with motherhood status. The small sample sizes for the earlier two KDHSs is a restriction but much more can be done with the much larger sample in the 2014 survey. A really interesting extension would be to make use of Q619 in this survey where women are asked to classify their most recent partner as casual or a regular boyfriend.\n\nThe distinction made between short-acting (pills, condoms) and long-acting (Injectables, implants, IUDs) is contentious. Many analysts prefer to classify injectables as short-acting because of high discontinuation. The 2014 KDHS indicates that the all-cause 12 month discontinuation probability for injectables is 31%, lower than pills or condoms at about 43% but much higher than implants or IUDs at 6-8%. You either need to change your definition or defend it.\n\nMethod choice is certainly of interest but the paper would benefit from an introductory table showing trends in use of any modern method among married or sexually active single women aged 15-24. The opening lines of the Discussion refer to a large increase in contraceptive use but your paper presents no data to support this.\n\nMinor points:\nIntroduction para 1: It is unclear what 20% means.\n\nIntroduction para 3: The mean age of 17 is misleading because it is based only on the minority who have given birth.\n\nIntroduction para 4: I do not understand what is meant by “The 15-24 year old women take about 15%...”\n\nIntroduction para 5: Who has claimed that the demographic window opens in 2038? Fertility has been falling in Kenya since the 1980s. You should also explain what is meant by the window.\n\nPage 3 penultimate para: Define Larcs here.\n\nMethods para1: The definition of current use is confusing.\n\nIs the work 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": "5629",
"date": "17 Jun 2020",
"name": "Margaret Kungu",
"role": "Author Response",
"response": "Thank you Prof. Cleland for the quick, expert, and very useful comments. I am sorry I did not respond earlier as I thought other reviewer comments would come soon after I respond together. I will address all the areas you have highlighted in order to give readers a quality paper. I am especially excited about adding the results of Q619/630 on the most recent sexual partner which will be very interesting for youth RH programs."
}
]
},
{
"id": "136459",
"date": "03 May 2022",
"name": "Jen Sothornwit",
"expertise": [
"Reviewer Expertise Family planning"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 secondary analysis from DHS. The study showed the trends in contraceptive methods as well as factors influencing the use in young women.\n\nIntroduction:\nIt was well written and easy to follow.\n\nRapid-repeat pregnancy is rather an old term. Short inter-pregnancy interval is a term suggested by society of family planning and other organizations.\n\nIntroduction lead to understanding why contraception is imperative. However, analysis was focusing on long- and short-term methods. It would be beneficial if the authors could provide more information on benefits, etc. regarding short- vs long term.\n\nMethods:\nWas multivariate analysis performed? If yes, provide more information on this analysis (esp. criteria for choosing variable into model, what method was used).\n\nIn my opinion, analysis of use vs non-use is important. I am not sure whether this meets the research objective or not.\n\nResults:\nWhy the authors did not provide the OR, adjusted OR for each variable? It would be more informative if they could.\n\nWas the third objective is \"utilizing long-term method\"?\n\nDiscussion:\nThe authors did a very good job discussing these results.\n\nImplication practice should move from the last paragraph in the conclusion section to the discussion. More references should be cited to support the proposed strategy.\n\nStrength and limitation should be provided.\n\nConclusion:\nSome sentences were not directly related to the study results and were over-extrapolated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "8190",
"date": "05 May 2022",
"name": "Margaret Kungu",
"role": "Author Response",
"response": "Thank you for taking the time to review and give very useful comments on the study. They are very much appreciated and will go a long way towards improving the study. We are reviewing the comments and will endeavor to revise the study guided by them and the comments of the first reviewer and we believe the study will be much better after that for a final print."
}
]
}
] | 1
|
https://f1000research.com/articles/9-197
|
https://f1000research.com/articles/9-193/v1
|
18 Mar 20
|
{
"type": "Research Article",
"title": "Determinants of contraceptive use among married women in Indonesia",
"authors": [
"Alfian Gafar",
"Dewi Elizadiani Suza",
"Ferry Efendi",
"Eka Mishbahatul Mar’ah Has",
"Ahmad Putro Pramono",
"Ika Adelia Susanti",
"Alfian Gafar",
"Dewi Elizadiani Suza",
"Eka Mishbahatul Mar’ah Has",
"Ahmad Putro Pramono",
"Ika Adelia Susanti"
],
"abstract": "Background: Contraceptives in family planning are used to control the timings between pregnancies. Although the number of those using family planning has increased, determinants of contraceptive use among married women in Indonesia remain insufficient. This research aimed to identify the factors associated with contraceptive use among married women in Indonesia. Methods: This study employed data from the Indonesian Demographic and Health Survey 2017. We selected 35,621 married women aged 15–49 years. Then, the determinants of contraceptive use among married women in Indonesia were examined by binary logistic regression. Results: Women’s age (Adjusted Odds Ratio (AOR)=0.529; 95% CI=0.470–0.597), the number of living children (AOR=44.024; 95% CI=33.193–58.390), education level (adjusted odds ratio=2.800; 95% CI=2.181–3.594), wealth index (AOR=1.104; 95% CI=0.978–1.246), frequency of watching television (AOR=1.555; 95% CI=1.321–1.829), and frequency of using the Internet (AOR=0.856; 95% CI=0.794–0.924) were significantly associated with contraceptive use among married women. Conclusions: This study highlights the determinants of contraceptive use among married women in Indonesia. Women’s age, the number of living children, education level, wealth index, and access to information may influence contraceptive use among these women. This study emphasizes that health education and promotion on the importance of using contraception should be initiated in innovative ways.",
"keywords": [
"Contraceptive",
"Demographic and Health Survey (DHS)",
"Determinants",
"Married Women"
],
"content": "Introduction\n\nFamily planning is a conscious effort made by couples to limit the number of children through the use of contraceptive methods. Both developed and developing countries worldwide have demonstrated increased contraceptive use to control the population1. Though contraceptives have been used globally, they remain less prevalent in poorer countries2. In Indonesia, the prevalence of contraceptive use among married women is still low and varies between provinces, economic status, education level, and residential location3. Based on data from the Indonesian health ministry, the percentage of contraception is divided into the following four categories: 59.3% of married women aged 15–49 years use modern contraception methods (implants, tubectomy, vasectomy, intrauterine devices, condoms, injections, and pills), 0.4% use traditional methods (lactation amenorrhea methods, periodic abstinence, and interrupted into intercourse), 24.7% have done family planning at least once, and 15.5% have never done family planning3–5. Although the number of those who actively use family planning has increased, it has not been able to sustain or increase the prevalence of contraceptive use6.\n\nNational data show that contraceptive use fell from 61.75% in 2014 to 59.98% in 20156. One of the challenges in increasing the number of family planning participants is the high level of concern from couples of reproductive age regarding the side effects of contraceptive drugs and equipment6. Researchers in Ghana have performed a study to identify the factors related to the low contraception use. They found that such factors included the residential location, knowledge, marital status, religion, and partner’s approval and support7. All efforts that can improve women’s health should be implemented, particularly regarding maternal healthcare services8. Thus, the current study aimed to identify factors that are associated with contraceptive use among married women in Indonesia.\n\n\nMethods\n\nThe data for this study were collected from the 2017 Indonesia Demographic and Health Survey (DHS), which is the eighth survey since 1987. The IDHS in 2017 was performed with the cooperation of the Central Statistics Agency, the National Population and Family Planning Agency, and the Ministry of Health, with technical assistance from the Inner City Fund (ICF) internationally through the Demographic Project and Health Surveys (DHS) Program. We used the individual recoded dataset in this study.\n\nOur sample was composed of married women aged 15–49 years who were using contraception in Indonesia. The survey successfully interviewed 49,627 women. Based on the inclusion criteria, 35,621 women remained. The IDHS used two-stage stratified cluster sampling to select the sample, including 1,970 census blocks covering urban and rural areas. The inclusion criteria in this study were all married women aged 15–49 years and who answered the questionnaire properly, whereas the exclusion criterion was women who were not married.\n\nThe dependent variable of this study was contraceptive use, which was defined in this study as the use of contraception by married women at the time of the survey. For the explanatory variables, we used the women’s age, the number of living children, education level, wealth index, residential location, the frequency of watching television, and access to the Internet.\n\nThe data were analyzed using the STATA statistical software version 14. Before the analysis, the dataset was weighted to account for any differences, considering the nature of the sampling design method. Univariate analysis and bivariate analysis were performed using the chi-square test. Finally, the determinants of contraceptive use among married women were identified using the binary logistic regression.\n\nEthical review and consent\n\nThe IDHS in 2017 obtained ethical permits from the Ministry of Health of Indonesia. All respondent identifiers were deleted from the data, and written informed consents were provided by each participant. The ICF International, which is part of the DHS program, approved the use of such data in this study.\n\n\nResults\n\nMore than half of the respondents used contraception (63.60%) in which majority of them aged 35–49 years (54.77%). According to the data, contraceptive use was most prevalent in married women who had 1–2 children (62.58%). Furthermore, most of the respondents had completed secondary education (52.03%) and were classified as richer by the wealth index (21.22%). Meanwhile, slightly more than half of them were from rural areas (51.62%). Regarding information access, 34,367 (96.48%) relied on watching television, whereas 12,733 (35.74%) relied on the Internet. Data are presented in Table 1.\n\nIn the bivariate analysis, all of the variables, except for the residential location, were significantly associated with contraceptive use among married women (Table 2).\n\n*p-value<0.05; **p-value<0.01: ***p-value<0.001\n\nIn the multivariate analysis, the association between the independent and dependent variables was assessed by binary logistic regression (Table 3). Married women aged 35–49 years were less likely to use contraceptives than married women aged 15–24 years (Adjusted Odds Ratio (AOR) =0.529; 95% CI=0.470–0.597). Furthermore, married women with more than five children were more likely to use contraception than those without children (AOR=44.024; 95% CI=33.193–58.390). Women who had completed higher education had 2.8 times greater odds of using contraceptives (AOR=2.800; 95% CI=2.181–3.594) than those who had not completed any formal education. Women classified as the richest by the wealth index were 1.1 times more likely to use contraceptives than those classified as poorest (AOR=1.104; 95% CI=0.978–1.246). Women who watched television were more likely to use contraceptives (AOR=1.555; 95% CI=1.321–1.829) than those who never watched television. In comparison, married women who accessed information from the Internet were less likely to use contraceptives (AOR=0.856; 95% CI=0.794–0.924).\n\n*p-value<0.05; **p-value<0.01: ***p-value<0.001\n\n\nDiscussion\n\nThis study sought to assess the determinants of contraceptive use among married women in Indonesia. We first found that women’s age, especially older age, was significantly associated with contraceptive use among married women. This result is consistent with the previous studies conducted in Ghana and Nigeria, documenting that older women have a lower level of concern with modern contraceptive use7,9. This finding is related to their lower fecundity rates and less active sexual desires10. The benefits of using contraception are to delay or space subsequent pregnancies and to limit the number of children2,11,12. Although older women use contraception less often than young women, they still considered the use of contraceptives; of note, only a small proportion of women aged 35–49 reported having gone through menopause.\n\nWomen with five or more living children were more likely to use contraceptives. This finding is consistent with those of studies performed in Ghana7,13. The addition of one child will increase the tendency of married women to use contraception by 7%–8%. Another study also found that women with more than three living children were more likely to use contraceptives than those without children, and their reason of using contraceptives was to prevent from having more children9,10,14,15. Women will choose to use contraception when they have reached their ideal family size7. Therefore, women who have many children are more likely to use contraception because they are more likely to have reached their ideal family size. The World Health Organization has reported that maternal mortality is increased in women who have more than four children1. Multiparous women have the highest risk of maternal death; therefore, contraceptive use should be encouraged to reduce this mortality16.\n\nMoreover, education levels were significantly associated with contraceptive use among these married women. Higher education levels correlated with higher use of contraceptives. Two previous studies conducted in Bangladesh and Ghana reported that education has an extremely significant influence on contraceptive use. In this report, women with higher education were more likely to use contraception than those without formal education14,15. This finding is the result of highly educated people being more likely to be aware of the benefits and importance of using contraception14. Another study in Nigeria also found that educated women were more likely to use contraceptives9. Education is commonly assumed by someone’s knowledge. Someone with higher education level has better knowledge. Education remains an important factor in terms of increasing women’s knowledge regarding family planning.\n\nWealth index was also significantly associated with contraceptive use among these married women. Women from the richest tier of the wealth index also had odds that progressively increased to using a contraceptive. Similar findings regarding the strong association between wealth index and contraceptive use were obtained by studies conducted in Malawi and Ghana; women classified within the richest wealth index were more likely to use contraception than those grouped in the poorest index7,9,15,17. In 2017, Ofonime stated that financial factors play an important role in the decreased use of contraceptives among the poorest married women18. Providing free access for contraceptives to poor women would be beneficial to increase the use of contraceptives.\n\nMeanwhile, access to information showed a significant association with contraceptive use among these married women. Mubashar et al. in 2016 found that most sources of contraception information were from media, such as television, radio, the Internet, local news, newspaper, and magazine19. Access to information from television was significantly associated with contraceptive use. Women who watched television had approximately 1.5 greater odds of using contraceptives than those who never watch television. This finding is consistent with the previous study conducted in Ethiopia and Ghana20–22. Exposure to media, such as television, is an important factor related to women’s knowledge about contraceptive use20. Watching television is important to increase the knowledge of the wider community to understand the types, benefits, and methods of using contraception correctly. In contrast, this study also showed that access to information via the Internet was significantly associated with less likelihood of contraceptive use among married women. This finding is supported by a previous study conducted in Australia23. In such study, 50% of women reported that they felt dissatisfied with the quality and quantity of information about contraception, especially oral contraceptive pill, they obtained through the Internet. Improving the sources of information about contraceptives for married women will be the best approach to encourage contraceptive use and avoid bias information from unreliable media sources.\n\n\nConclusions\n\nFactors such as women’s age, the number of living children, education level, wealth index, and access to information remain significant issues in determining contraceptive use among married women in Indonesia. Overall, the study results suggest that policymakers should target certain women and create a campaign regarding contraception. Targeting older, poor, and uneducated or less educated women may have a positive impact in terms of increasing their use of contraception.\n\n\nData availability\n\nData used in this study is available online from the Indonesian 2017 Demographic and Health Survey (DHS) website under the ‘Individual Recode’ section.\n\nData can be accessed by applying through the DHS website. Please see their data access help page for information.",
"appendix": "References\n\nWHO: Family planning/Contraception. WHO. Published 2019. Reference Source\n\nUnited Nations, Department of Economic and Social Affairs, Population Division: Trends in Contraceptive Use Worldwide 2015. (ST/ESA/SER.A/349). 2015. Reference Source\n\nBPS, BKKBN, Kemenkes, ICF: Survei Demografi Dan Kesehatan Indonesia 2012. Jakarta; 2012. Reference Source\n\nBadan Penelitian dan Pengembangan Kesehatan: Riset Kesehatan Dasar 2013. Ris Kesehat Dasar 2013. 2013. Reference Source\n\nKementerian Kesehatan: Situasi dan Analisis Keluarga Berencana. Kementeri Kesehat Republik Indones. 2014; 2. Reference Source\n\nPPN/Bappenas K: Evaluasi Paruh Waktu RPJMN 2015-2019. 2017; 211. Reference Source\n\nAviisah PA, Dery S, Atsu BK, et al.: Modern contraceptive use among women of reproductive age in Ghana: analysis of the 2003-2014 Ghana Demographic and Health Surveys. BMC Womens Health. 2018; 18(1): 141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurniati A, Chen CM, Efendi F, et al.: Factors influencing Indonesian women's use of maternal health care services. Health Care Women Int. 2018; 39(1): 3–18. PubMed Abstract | Publisher Full Text\n\nOluwasanu MM, John-akinola YO, Desmennu AT, et al.: Access to Information on Family Planning and Use of Modern Contraceptives Among Married Igbo Women in Southeast, Nigeria. Int Q Community Health Educ. 2019; 39(4): 233–243. PubMed Abstract | Publisher Full Text\n\nWai MM, Bjertness E, Htay TT, et al.: Dynamics of contraceptive use among married women in North and South Yangon, Myanmar: findings from a cross-sectional household survey. Contracept X. 2020; 2: 100015. Publisher Full Text\n\nAl Kindi RM, Al Sumri HH: Prevalence and sociodemographic determinants of contraceptive use among women in Oman. East Mediterr Health J. 2019; 25(7): 495–502. PubMed Abstract | Publisher Full Text\n\nAlsaleem MA, Khalil SN, Siddiqui AF, et al.: Contraceptive use as limiters and spacers among women of reproductive age in southwestern, Saudi Arabia. Saudi Med J. 2018; 39(11): 1109–1115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNketiah-Amponsah E, Arthur E, Aaron A: Correlates of Contraceptive use among Ghanaian women of Reproductive Age (15-49 Years). 2012; 16(3): 155–170. PubMed Abstract\n\nIslam AZ, Mondal MN, Khatun ML, et al.: Prevalence and Determinants of Contraceptive use among Employed and Unemployed Women in Bangladesh. Int J MCH AIDS. 2016; 5(2): 92–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdebowale SA, Adedini SA, Ibisomi LD, et al.: Differential effect of wealth quintile on modern contraceptive use and fertility: evidence from Malawian women. BMC Womens Health. 2014; 14(1): 40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGanatra B, Faundes A: Role of birth spacing, family planning services, safe abortion services and post-abortion care in reducing maternal mortality. Best Pract Res Clin Obstet Gynaecol. 2016; 36: 145–155. PubMed Abstract | Publisher Full Text\n\nNyarko SH: Prevalence and correlates of contraceptive use among female adolescents in Ghana. BMC Womens Health. 2015; 15: 60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson OE: Determinants of Modern Contraceptive Uptake among Nigerian Women: Evidence from the National Demographic and Health Survey. Afr J Reprod Health. 2017; 21(3): 89–95. PubMed Abstract | Publisher Full Text\n\nMubashar H, Almushait MA, Sukit B, et al.: Knowledge, attitude and practice of contraceptives among Saudi women in Aseer Region, Saudi Arabia. Bangladesh J Med Sci. 2016; 15(3): 430–434. Publisher Full Text\n\nMedhanyie AA, Desta A, Alemayehu M, et al.: Factors associated with contraceptive use in Tigray, North Ethiopia. Reprod Health. 2017; 14(1): 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeson P, Appiah R, Adomah-Afari A: Modern contraceptive use among reproductive-aged women in Ghana: prevalence, predictors, and policy implications. BMC Womens Health. 2018; 18(1): 157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTekelab T, Melka AS, Wirtu D: Predictors of modern contraceptive methods use among married women of reproductive age groups in Western Ethiopia: a community based cross-sectional study. BMC Womens Health. 2015; 15: 52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhilipson S, Wakefield CE, Kasparian NA: Women's knowledge, beliefs, and information needs in relation to the risks and benefits associated with use of the oral contraceptive pill. J Womens Health (Larchmt). 2011; 20(4): 635–642. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "61507",
"date": "24 Mar 2020",
"name": "Angeline Bushy",
"expertise": [
"Reviewer Expertise Rural health issues/nursing",
"Public Health Nursing",
"Qualitative Research Methodologies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is cogently organized with an appropriate quantitative methodology. The findings can provide insights to educators, researchers, as well as policymakers regarding family planning education and contraceptive needs and developing countries. I do not offer any editorial suggestions or revisions to the current manuscript. I recommend indexing without revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "5408",
"date": "08 Apr 2020",
"name": "Ferry Efendi",
"role": "Author Response",
"response": "Dear Reviewer, We would like to sincerely thanks the reviewer for reviewing our manuscript and the constructive comments."
}
]
},
{
"id": "61505",
"date": "06 Apr 2020",
"name": "Khatijah Binti Abdullah Lim Geok Khim",
"expertise": [
"Reviewer Expertise Women and Child Health",
"Critical Care",
"Management",
"Mixed methods",
"Qualitative 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 manuscript is well written with clear explanation of the methodology used. Data collected were analysed using appropriate statistical tests. Interpretations of the data were correctly displayed and discussed. Study findings highlight the determinants of contraceptive use among married women in Indonesia which can help in formulating relevant policy and strategies in improving contraceptive use in Indonesia. I have no recommendation for any revision to the manuscript in its current format and have no objection for it to be indexed with no revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "5409",
"date": "08 Apr 2020",
"name": "Ferry Efendi",
"role": "Author Response",
"response": "Dear Editor and Reviewer, We would like to sincerely thank you for considering our manuscript “Determinants of contraceptive use among married women in Indonesia” for F1000 research. We would also like to thank the reviewers for reviewing our manuscript and their constructive comments."
}
]
}
] | 1
|
https://f1000research.com/articles/9-193
|
https://f1000research.com/articles/9-192/v1
|
18 Mar 20
|
{
"type": "Research Article",
"title": "Determinants of preeclampsia and eclampsia among women delivering in county hospitals in Nairobi, Kenya",
"authors": [
"Gorbee G. Logan",
"Peter K. Njoroge",
"Lambert O. Nyabola",
"Marshal M. Mweu"
],
"abstract": "Background: Preeclampsia is defined as the onset of a new episode of high blood pressure in a woman usually after 20 weeks gestation plus proteinuria, whereas eclampsia is defined as generalized seizures in a pregnant woman who generally has preeclampsia criteria. Preeclampsia and eclampsia are hypertensive disorders of pregnancy and thus, among the top causes of maternal death worldwide. The objective of this study was to investigate risk factors for preeclampsia/eclampsia. Methods: This was a hospital-based unmatched case-control study carried out among women of reproductive age (15-49 years) who have given birth at Nairobi County Hospitals and admitted to the postnatal ward July-September 2019 with a sample size of 352 participants (88 cases and 264 controls). All cases were selected, while controls were simple random sampled, as per eligibility criteria. Information or data were gathered using a structured interviewer-administered questionnaire and data abstraction tool. Descriptive analysis was carried out, where, categorical variables were presented in percentages or proportions, whereas; continuous variables were presented in means, standard deviations, and range. This was followed by a bivariable mixed-effect logistic regression analysis and a multivariable mixed-effect logistic regression analysis using the significant variables from bivariable analysis. Results: Of all the 88 cases enrolled in the study 5 (5.68%) had eclampsia and 83 (94.32%) had preeclampsia. There was a significant association between personal history of hypertension (AOR=7.1; 95% CI: 2.6-19.3, p=0.001), Occupation as a housewife (AOR=3.1; 95% CI: 1.1-8.8, p=0.034), nulliparity (AOR=7.5; 95% CI: 1.5-37.5, p=0.015) , primiparity (AOR=2.1; 95% CI: 1.1-4.2, p=0.031), advanced maternal age 35-49 years (AOR=5.9; 95% CI: 1.1-33.3, p=0.042), and the occurrence of preeclampsia/eclampsia. Conclusions: The following conclusions were made regarding the study findings: Personal history of hypertension, older/advanced maternal age (35-49 years), occupation, and parity were factors significantly associated with preeclampsia/eclampsia.",
"keywords": [
"Preeclampsia",
"Eclampsia",
"Determinants"
],
"content": "Introduction\n\nPreeclampsia affects 2–10% of pregnant women globally and eclampsia 0.03–0.05%1. However, the overall prevalence of preeclampsia according to studies varies from 4.5% to 23%2. Preeclampsia affects about 4% of pregnancies in the United States of America3. In Kenya, the incidence of preeclampsia is about 0.3%4 while the prevalence is 6.1%5. Similarly in Ethiopia, the prevalence of preeclampsia is about 5%6. In Nigeria, the prevalence of preeclampsia ranges between 2 and 16.7%7.\n\nThe maternal mortality ratio in Sub-Saharan Africa is estimated to be 510 maternal deaths per 100,00 live births8. Maternal and fetal complications in addition to maternal and fetal mortality are much greater in mothers with pre-eclampsia and eclampsia than those without2. Preeclampsia is the second top cause of maternal death globally, which may lead to grave maternal complications (stroke, eclampsia, and organ failure), and poor perinatal outcome for the fetus and infant especially intrauterine development restriction, low birth weight, and stillbirth9. Preeclampsia develops in 20% of first pregnancies and entails more than 40% of premature birth resulting from treatment2. About 98% of maternal, fetal, and neonatal death related to preeclampsia and eclampsia occur in low-income countries, with much of this mortality in South Asia and Sub-Saharan Africa10. Preeclampsia also accounts for increasing maternal & perinatal/infant mortality, and it is a major cause of maternal mortality 15–20% in developed countries11.\n\nPreeclampsia and eclampsia are among the hypertensive disorders of pregnancy. About 13% of maternal mortality globally is due to hypertensive disorders of pregnancy and the number is even higher in developing countries with an estimate between 20–80% in Africa and Latin America1.\n\nMany risk factors have been associated with preeclampsia/eclampsia2. Nevertheless, a full account of the risk factors of preeclampsia/eclampsia has not been well established in the Kenyan population4. Therefore, this study was conducted to investigate some risk factors of preeclampsia/eclampsia namely; socio-demographic, reproductive and obstetric, clinical, behavioral and family history-related factors, with a view of informing policy, creating awareness, and formulating strategies to improve antenatal care and delivery services among women of reproductive age in Kenya.\n\n\nMethods\n\nThis was a hospital-based unmatched case-control study carried out at Nairobi County Hospitals’ postnatal ward. This study design was chosen because it is appropriate for identifying risk factors associated with pre-eclampsia/eclampsia, which are latent conditions among gravid women.\n\nThis study was carried out at Mbagathi District Hospital (Kibra), Mama Lucy Kibaki Hospital (Embakasi Central), and Pumwani Maternity Hospital (Kamukunji) in Nairobi County, Kenya; with a catchment population of 4.6 million people, 58% of which are slum dwellers. These are public county hospitals offering primary, secondary, and tertiary health care services to Nairobi residents and neighboring counties such as Kajiado, Kiambu and Machakos counties.\n\nThe study population was women of reproductive age (15–49 years) who have given birth at Nairobi County Hospitals and admitted to the postnatal ward from July–September, 2019. The controls and cases were selected from the study population-based outlined criteria for eligibility. A postnatal ward mother that met/satisfied the definition of a case or control and consented to participation was included. Those aged less than 18 years old were included in the study, but both the parent/guardian and patient were required to sign consent and assent forms, respectively. A postnatal ward mother that did not meet the definition of a case or control was excluded from the study.\n\nA case was defined as a woman of reproductive age (15–49 years) admitted in postnatal wards having been diagnosed of preeclampsia (high blood pressure during pregnancy, ≥140/90 mmHg, plus proteinuria ≥300 mg/24 hours or ≥ 1+ dipstick) or eclampsia (high blood pressure during pregnancy, proteinuria plus generalized seizures) at Nairobi County Hospitals July- September, 2019. All cases that met the case definition were selected during the study period, July-September 2019, to obtain the required sample of 88 cases and the response rate was 100%. Thus, each case was selected after the physician (medical officer or consultant obstetrician/gynecologist) had made a diagnosis (preeclampsia/eclampsia) and client admitted to the postnatal ward.\n\nControl was defined as a woman of reproductive age (15–49 years) admitted to the postnatal ward without preeclampsia/eclampsia at Nairobi County Hospitals from July-September 2019. Controls were simple randomly sampled from a list of women admitted to each of the three hospitals' post-natal ward without preeclampsia/eclampsia which was recruited at the time of selection of the cases. A total of three controls for each case were selected by the time of selection to cases. The sampling frame was updated as per deliveries taking place in study hospitals from July-September 2019.\n\nThe sample size of 352 participants (264 controls & 88 cases) was determined as specified by Kelsey, Jennifer, and others12 for case-control studies as follow:\n\n\n\n\n\n\n\n\n\n\n\nSince the outcome is rare in the Kenyan population, the ratio of 3 controls to 1 case was used in calculating the sample size but still maintains the statistical power of the study; N1 is the number of cases and N2 is the number of controls. Furthermore, P1 is the proportion of cases exposed thus, was the proportion of women with pre-eclampsia/eclampsia that attend less than four antenatal clinic (ANC) visits; P2 is the proportion of controls exposed and this was the proportion of women without pre-eclampsia/eclampsia that attends less than four ANC visit, that was set at 42%13; Zα (1.96) and Zβ (-0.84) were the required values specifying the two-tailed confidence interval (95%) and statistical power (80%) desired respectively. The odds for attending less than four ANC visits-preeclampsia/eclampsia association was been set at 2 (universally acceptable) and r=3, is the ratio of controls to cases. Given these figures, the desired sample size of 352 participants (88 cases and 264 controls) was computed.\n\nThe dependent variable was pre-eclampsia/eclampsia status measured as a binary categorical variable. The independent variables of interest were maternal age, education, residence, marital status, occupation, ethnicity, religion, maternal age, age at first marriage, age at first pregnancy, number of antenatal care visit, time/trimester of first ANC visit, gravidity, parity, child sex, anaemia in pregnancy, urinary tract infection (UTI) in pregnancy, alcohol use, tobacco use, family history of hypertension, family history of diabetes, personal history of diabetes, personal history of hypertension, and use of traditional treatment or medicine. All of these explanatory variables were more or less potential confounders and a determination of which one is a confounder was made during analysis. A pretested structured interview questionnaire was used to collect primary data from postnatal mothers and a data abstraction tool was also used to collect some secondary data, both available as Extended data14.\n\nThe research was granted ethical clearance by Kenyatta National Hospital (KNH)-University of Nairobi (UON) Ethics and Research Committee (P426/05/2019) and the Nairobi City County Government - Public health division (Ref. No. NCC/HRD/HRM/11/904/JWN/2019). Furthermore, written consent was obtained from the participants after they were informed about the nature of the study.\n\nBefore starting data collection, six research assistants (two for each of the three health facilities) were trained on the sampling technique for cases and controls and standard interview skills to reduce systemic error and interview bias. Research assistants were also trained to get information such as the number of antenatal care (ANC) visits, time/trimester of first ANC visit from the patient medical record using data abstraction tool in an attempt to reduce recall bias. Misclassification bias which could affect both cases and controls was minimized by including cases and controls determined by the obstetrician.\n\nThe filled questionnaires were first checked for completeness then followed by the entry of data collected from the field into Microsoft Excel spreadsheets, cleaned, formatted, coded and audited for quality and consistency using Epi-info software before exporting the dataset to Stata -version 14.0 computer programming software. Descriptive analysis was carried out, where, categorical variables were presented in percentages or proportions, whereas, continuous variables were presented in means and standard deviations. This was followed by bivariable mixed-effect logistic regression analysis and a multivariable mixed-effect logistic regression analysis using the significant variables (p ≤0.20) from the bivariable analysis. A modified Hosmer-Lemeshow goodness-of-fit test was carried out; after which, variables with p≤0.05 were considered as factors associated with preeclampsia/eclampsia.\n\n\nResults and discussion\n\nSocio-demographic characteristics of the study participants. The study participants were 352, of whom 88 were cases and 264 were controls. Among the cases, preeclampsia accounted for 83 (94.3%) and eclampsia 5 (5.7%). This was a multicenter study conducted in Pumwani Maternity Hospital which had 196 participants (55.7%), Mama Lucy Kibaki Hospital with 99 participants (28.1%) and Mbagathi District Hospital with 57 participants (16.2%). The distribution of study participants by study hospital is presented in Table 1; individual-level responses to questionnaire items are available as Underlying data\n\nThe mean maternal age of the study participants was 26.1 years (SD=5.5); the mean maternal age of controls being 26.1 years with a standard deviation 5.3 years (range: 18–41 years) and the mean maternal age of cases being 27.6 years with 5.9 standard deviation (range: 17–42). About 81% of cases and 83% of controls were mothers aged between 20 and 34 years old. Among the controls and cases, 71 (26.9%) and 23 (26.1%) had primary-level education, respectively, whereas 140 (53.0%) of the controls and 40 (45.5%) of cases had a secondary level of education. In terms of marital status, 80.7% of cases and 80.7% of controls were married. The majority of cases 49 (55.7%) and controls 114 (43.2%) had an occupation as housewife.\n\nConcerning the county of residence, 247 (93.6%) of controls and 86 (97.7%) of cases were residents of Nairobi County. Mothers of Kenyan ethnicity accounted for 258 (97.7%) of controls and 86 (97.7%) of cases whereas, those of non-Kenyan ethnicity were 2 (2.3%) of cases and 6 (2.3%) of controls. Among the cases and controls, 97.7% and 97.0% were Christians respectively; whereas Muslim mothers represented 8 (3.0%) controls and 2 (2.3%) of cases. The socio-demographic factors of the study population are presented in Table 2.\n\nThe socio-demographic factors associated with preeclampsia/eclampsia. The socio-demographic factors hypothesized to significantly associate with preeclampsia/eclampsia include maternal age, maternal level of education, marital status, maternal occupation, maternal county of residence, and religion.\n\nCompared to mothers aged less than 20 years, mothers 20–34 years of age were 1.5 times more likely to suffer preeclampsia/eclampsia (OR=1.5, 95% CI=0.5-4.7, p=0.451) whereas mothers aged 35–49 years were 2.5 times more likely to experience preeclampsia/eclampsia than mothers aged less than 20 years (OR=2.5, 95% CI=0.7-8.8, p=0.163).\n\nPostnatal mothers from government/private occupation were 1.8 times more likely to suffer from preeclampsia/eclampsia than those from other occupations (OR=1.8, 95% CI=0.6-5.4, p=0.286) whereas those that have an occupation as housewives were 2.0 times more likely to develop preeclampsia/eclampsia than other occupations. Similarly, mothers that have merchant/business occupation were more likely to develop preeclampsia/eclampsia than other occupations (OR=1.1, 95% CI=0.5-2.9, p=0.789).\n\nPostnatal mothers from Nairobi County were at increased risk of preeclampsia/eclampsia compared to those from other counties (OR=3.0, 95% CI=0.7-13.1, p=0.152). The associations between socio-demographic factors and preeclampsia/eclampsia are summarized in Table 3.\n\nThe reproductive and obstetric factors associated with preeclampsia/eclampsia\n\nWe hypothesized that reproductive and obstetrics factors likely associated with preeclampsia/eclampsia were maternal age, age at first marriage, age at first pregnancy, number of ANC visits, time/trimester of first ANC visit, gravidity, parity, and child-Sex.\n\nNulliparous mothers were 4.8 times more likely to suffer from preeclampsia/eclampsia than multiparous mothers (OR=4.8, 95% CI=1.0-22.4, p=0.045) whereas primiparous mothers were 1.4 times more likely to develop preeclampsia/eclampsia than those that were multiparous (OR=1.4, 95% CI=0.9-2.3, p=0.187). Nulliparous mothers, though, were at increased risk of suffering preeclampsia/eclampsia; however, the association was not statistically significant in that the 95% confidence included 1 despite a p-value of 0.045 (OR=4.8, 95% CI=1.0-22.4, p=0.045). Therefore, there was no significant association between parity and development of preeclampsia and or eclampsia. Compared to mothers aged 20 years and above, teenage mothers were 30% less likely to develop preeclampsia/eclampsia (OR=0.7, 95% CI=0.4-1.3, p=0.199).\n\nCompared to mothers whose first antenatal care visit was in the first trimester of pregnancy, mothers whose first antenatal care visit was in the second and third trimesters were at reduced risk of preeclampsia/eclampsia (OR=0.8, 95% CI=0.5-1.4, p=0.473 and OR=0.5, 95% CI=0.2-1.4, p=0.191). Mothers whose age at first marriage was <20 years old were 10% less likely to develop preeclampsia/eclampsia than those that were ≥20 years old (OR=0.9, 95% CI=0.5-1.6, p=0.659) whereas mothers whose age at first pregnancy was <20 years old were 0.7 times more likely to develop preeclampsia/eclampsia than those that were ≥20 years old (OR=0.7, 95% CI=0.4-1.3, p=0.199). The associations between reproductive and obstetric factors and preeclampsia/eclampsia are summarized in Table 4.\n\nThe clinical factors associated with preeclampsia/eclampsia\n\nAnemia and urinary tract infections in pregnancy were hypothesized to relate significantly with the occurrence of preeclampsia and eclampsia. Compared to mothers with a normal number of pus cells (0–5 hpf) on admission for delivery, mothers with mild-moderate and severe UTI (6–10 hpf and >10 hpf) in pregnancy on admission were 1.7 times more likely to develop preeclampsia/eclampsia (OR=1.7, 95% CI=0.4-7.0, p=0.454). Mothers with mild-moderate and severe levels (7-10.9 g/dl and <7 g/dl) of anemia on admission for delivery were 0.9 times as likely to suffer from preeclampsia/eclampsia than those with normal hemoglobin level (≥11 g/dl); (OR=0.9, 95% CI=0.5-1.6, p=0.625). Associations between clinical factors and preeclampsia/eclampsia are presented in Table 5.\n\nThe behavioural and family history-related factors associated with preeclampsia/eclampsia\n\nFurther, the relationships between alcohol use, tobacco use, traditional treatment use, diabetes, and hypertension with preeclampsia/eclampsia were assessed. History of diabetes and hypertension were personal and family history.\n\nPostnatal mothers who used tobacco were 3.9 times more likely to develop preeclampsia/eclampsia than those who did not use tobacco (OR=3.9, 95% CI=1.0-14.9, p=0.046). Postnatal mothers who used tobacco were at increased risk of suffering preeclampsia/eclampsia; however, the association was not statistically significant in that the 95% confidence included 1 despite a p-value of 0.046 (OR=3.9, 95% CI=1.0-14.9, p=0.046). On the other hand, mothers with a personal history of hypertension were 6.3 times more likely to suffer from preeclampsia/eclampsia than those without a personal history of hypertension (OR=6.3, 95% CI=2.7-14.8, p=0.001). In addition, there was a significant association between personal history of hypertension and development of preeclampsia/eclampsia (p<0.001). The associations between behavioural and family history-related factors and preeclampsia/eclampsia are presented in Table 6.\n\nMultivariable mixed-effect logistic regression on risk factors for preeclampsia/eclampsia. A multivariable mixed-effect logistic regression model was used to determine the effect of various socio-demographic, reproductive and obstetric, clinical, behavioral and family history-related factors. Only variables that reached p ≤0.2 from the bivariable analysis were moved forward for multivariable analysis. Those variables that are of interest from previous studies were also moved forward for multivariable analysis despite not meeting the stated threshold requirement.\n\nFrom the best model, mothers with advanced age 35–49 years were 5.9 times more likely to develop preeclampsia/eclampsia when compared to those aged less than 20 years (AOR=5.9, 95% CI=1.1-33.3, p=0.042). Mothers who had an occupation as housewife were significantly more likely to suffer from preeclampsia/eclampsia compared to those with other occupations (AOR = 3.1; 95% CI: 1.1-8.8, p=0.034).\n\nMothers that had less than four ANC visits were 1.8 times more likely to suffer preeclampsia/eclampsia; however, the association was not statistically significant in that the 95% confidence included 1 despite a p-value of 0.041 (AOR=1.8, 95% CI=1.0-3.3, p=0.041). Compared to mothers who were multiparous, nulliparous and primiparous were significantly associated with increased risk of developing preeclampsia/eclampsia (AOR=7.5, 95% CI=1.5-37.5, p=0.015) and (AOR=2.1, 95% CI=1.1-4.2, p=0.031) respectively.\n\nMothers with preeclampsia/eclampsia were 7.1 times more likely to have personal history of hypertension comparing to their counterparts (AOR=7.1, 95% CI=2.6-19.3, p=0.001). The associations between risk factors and preeclampsia/eclampsia are presented in Table 7.\n\nWomen with preeclampsia/eclampsia were more likely to be 35–49 years of age comparing to their counterparts (AOR=5.9, 95% CI=1.1-33.3, p=0.042). This was concurrent with other studies that showed an increased risk in a similar age group11,15,16. A study conducted in Iran has linked the obstetric danger of advanced maternal age to aging-mediated vascular damage2. This is because as a woman gets advanced age, she is more likely to develop heart/blood vessel related problems chiefly due to the steady failure of compliance of said vessels that are mainly linked with the ageing of uterine blood vessels and arterial firmness. Thus, the reason why preeclampsia/eclampsia is more likely in older women is biologically plausible.\n\nThe odds of developing preeclampsia/eclampsia were 3.1 times higher in postnatal mothers with occupation as housewife comparing to mothers with other occupations (AOR=3.1, 95% CI=1.1-8.8, p=0.034). The association between occupation as housewife and preeclampsia/eclampsia was statistically significant. A similar finding was obtained from a study done in Nigeria17. This could be due to stress-related factors arising from low socio-economic status and low wealth index.\n\nAccording to the results of our study, women who were nulliparous or primiparous were at increased risk of developing preeclampsia/eclampsia than those who were multiparous. This was concurrent with several studies that found the same findings1,11,18. Several hypotheses have linked nulliparity to a maternal immune maladaptation2. This is for the reason that nulliparity is due to early trophoblastic invasion and how the mother reacts to it. The breakdown or malfunction of the normal invasion of trophoblastic cells leads to maladaptation of the coiled arterioles, which are linked to the causation of preeclampsia1.\n\nMothers that attended less than four ANC visits were at increased risk of developing preeclampsia/eclampsia when compared with those that attended four or more ANC visits. This is comparable to a study conducted in Nigeria17. Long-distance to access health facility, limited knowledge about antenatal care (ANC) services coupled with low socio-economic status could possibly be responsible for postnatal mothers not meeting the WHO recommended four or more ANC visits; a situation which could predispose mothers to obstetric complications such as preeclampsia and eclampsia. In this study however, the association between a number of ANC visits and the outcome (preeclampsia/eclampsia) was not statistically significant in that the 95% confidence included 1 despite a p-value of 0.041 (AOR = 1.8; 95% CI: 1.0-3.3, p=0.041).\n\nThe study results found out that anemia and UTI in pregnancy were not significantly associated with preeclampsia/eclampsia; however, this finding contradicts studies conducted in Egypt, Sudan, and by WHO19–21.\n\nWomen with personal history of hypertension were more likely to suffer from preeclampsia/eclampsia compared with their counterparts (AOR=7.1, 95% CI=2.6-19.3, p=0.001). A similar finding was found in a study conducted in Nigeria17. It is probable that lifestyle modifications/behavioral factors are the reason for influencing women to an increased threat of preeclampsia/eclampsia. For instance, mothers taking-in an unhealthy diet; eating food high in fats and carbohydrates could increase their triglyceride levels, narrowing blood flow, which may predispose them to develop hypertensive disorders in pregnancy.\n\nLimitations of the study. This study had some limitations. First, not all admission for delivery had their hemoglobin level (337 out of 352 study participants) and urinary pus cells count (only 66 out of 352) recorded in the study hospitals, therefore; if this study was repeated in other facilities with better recording of lab results of maternal admission, then the findings could be different. Secondly, there might have been recall bias regarding some factors such as the specific traditional treatment use and purpose of using the said treatment. Lastly, the hospital-based approach included only women attending the study hospitals.\n\n\nConclusions\n\nThis study indicated that a personal history of hypertension, older/advanced maternal age (35–49 years), occupation, and parity (nulliparous/primiparous) were factors significantly associated with preeclampsia/eclampsia. In this study, only 66 out of 352 participants (18.8%) had their urinary pus cells recorded on admission for delivery and whether UTI in pregnancy is actually not associated with preeclampsia/eclampsia needs further investigation. Anemia in pregnancy was 10% less likely to develop preeclampsia/eclampsia.\n\nBased on the study findings, this study makes the following recommendations to policymakers, county government, hospital management teams, and other relevant institutions:\n\n1. Health Workers in Maternal and Child Health units of health facilities should emphasize the risk factors for preeclampsia and/or eclampsia to pregnant and postnatal mothers during their health talks in the health facilities. These messages should be extended to other pregnant and postnatal mothers in the catchment areas of the health facilities through Community Health Workers.\n\n2. Maternity In-charges should ensure that urinary pus cell count and hemoglobin level for pregnant women are ordered and results recorded in the admission notes for delivery.\n\n3. That future studies should investigate the association between UTI in pregnancy and preeclampsia/eclampsia in a multi-county study.\n\n\nData availability\n\nHarvard Dataverse: Replication data for: Determinants of preeclampsia and eclampsia among women delivering in county hospitals in Nairobi, Kenya: https://doi.org/10.7910/DVN/BYFL3J14.\n\nThis project contains the following underlying data:\n\nLogan preeclampsia dataset Final. (Study dataset)\n\nLogan preeclampsia dofile Final2 (do file code for Determinants-Preeclampsia/ eclampsia identification).\n\nHarvard Dataverse: Replication data for: Determinants of preeclampsia and eclampsia among women delivering in county hospitals in Nairobi, Kenya: https://doi.org/10.7910/DVN/BYFL3J14.\n\nThis project contains the following extended data:\n\nLogan Interview Questionnaire & Data Abstarction Tool. (Questionnaire and data abstraction tool used in this study.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nWe would like to acknowledge and thank the School of Public Health, College of Health Sciences/University of Nairobi for providing all assistance requested. Special thanks go to the members of the Kenyatta National Hospital-University of Nairobi Ethics and Research Committee and the Nairobi City County Government – Public Health Division for granting me the approval to conduct this research. We are indebted to the management and staff of Pumwani, Mama Lucy Kibaki and Mbagathi hospitals for allowing us to conduct this study within their facilities. Our appreciation goes to the Government of Liberia (GOL), the Republic of Liberia for funding this research and the entire study in Kenya.\n\nLastly, we wish to acknowledge and thank all the postnatal mothers who participated in this study. Their excitement and willingness to fill the questionnaires made the completion of this research an enjoyable experience. Furthermore, special thanks to the research assistants and data entry clerks who helped us in collecting and entering data.\n\n\nReferences\n\nWandabwa J, Doyle P, Kiondo P, et al.: Risk factors for severe pre-eclampsia and eclampsia in Mulago Hospital, Kampala, Uganda. East Afr Med J. 2010; 87(10): 415–24. PubMed Abstract\n\nKahnamouei-aghdam F, Amani F, Hamidimoghaddam S: Prevalence of pre-eclampsia and eclampsia risk factors among pregnant women, 2011-2013. Int J Adv Med. 2015; 2(2): 128–32. Publisher Full Text\n\nUS Preventive Services Task Force, Bibbins-Domingo K, Grossman DC, et al.: Screening for Preeclampsia: US Preventive Services Task Force Recommendation Statement. JAMA. 2017; 317(16): 1661–1667. PubMed Abstract | Publisher Full Text\n\nItoh M, Were F, Owidhi M, et al.: Incidence of Pregnancy-Associated Hypertension and Preeclampsia in a cohort of Kenyan Women. Annals of Gynecology and Obstetrics. 2017; 1(1): 3–4. Publisher Full Text\n\nOtieno DAN: FACTORS CONTIBUTING TO ADVERSE OUTCOMES OF PRE-ECLAMPSIA AMONG PREGNANT WOMEN ATTENDING ANTENATAL CLINICS IN KIBERA SLUMS, NAIROBI. 2012. Reference Source\n\nWagnew M, Dessalegn M, Worku A, et al.: Trends of preeclampsia/eclampsia and maternal and neonatal outcomes among women delivering in addis ababa selected government hospitals, Ethiopia: a retrospective cross-sectional study. Pan Afr Med J. 2016; 25(Suppl 2): 12. PubMed Abstract | Free Full Text\n\nOsungbade KO, Ige OK: Public health perspectives of preeclampsia in developing countries: implication for health system strengthening. J Pregnancy. 2011; 2011: 481095. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKirigia JM, Muthuri RD, Nabyonga-Orem J, et al.: Counting the cost of child mortality in the World Health Organization African region. BMC Public Health. 2015; 15(1): 1103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUS Preventive Services Task Force, Grossman DC, Bibbins-Domingo K, et al.: Behavioral Counseling to Promote a Healthful Diet and Physical Activity for Cardiovascular Disease Prevention in Adults Without Cardiovascular Risk Factors: US Preventive Services Task Force Recommendation Statement. JAMA. 2017; 318(2): 167–174. PubMed Abstract | Publisher Full Text\n\nGoldenberg RL, Jones B, Griffin JB, et al.: Reducing maternal mortality from preeclampsia and eclampsia in low-resource countries--what should work? Acta Obstet Gynecol Scand. 2015; 94(2): 148–155. PubMed Abstract | Publisher Full Text\n\nShamsi U, Saleem S, Nishter N: Epidemiology and risk factors of preeclampsia; an overview of observational studies. Al Ameen J Med Sci. 2013; 6(4): 292–300. Reference Source\n\nKelsey JL, Whittemore AS, Evans AS, et al.: Methods in observational epidemiology. Monographs in Epidemiology and Biostatistics. 1996. Reference Source\n\nDemographic K: Health Survey (2014 KDHS). 2014. Reference Source\n\nLogan G: Replication Data for: Determinants of preeclampsia and eclampsia among women delivering in county hospitals in Nairobi, Kenya. Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/BYFL3J\n\nTessema GA, Tekeste A, Ayele TA: Preeclampsia and associated factors among pregnant women attending antenatal care in Dessie referral hospital, Northeast Ethiopia: a hospital-based study. BMC Pregnancy Childbirth. 2015; 15(1): 73. Publisher Full Text\n\nWikström AK, Stephansson O, Cnattingius S: Tobacco use during pregnancy and preeclampsia risk: effects of cigarette smoking and snuff. Hypertension. 2010; 55(5): 1254–1259. PubMed Abstract | Publisher Full Text\n\nGuerrier G, Oluyide B, Keramarou M, et al.: Factors associated with severe preeclampsia and eclampsia in Jahun, Nigeria. Int J Womens Health. 2013; 5: 509–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrum T, Seifu A, Abay M, et al.: Determinants of pre-eclampsia/Eclampsia among women attending delivery Services in Selected Public Hospitals of Addis Ababa, Ethiopia: a case control study. BMC Pregnancy Childbirth. 2017; 17(1): 307. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli AA, Rayis DA, Abdallah TM, et al.: Severe anaemia is associated with a higher risk for preeclampsia and poor perinatal outcomes in Kassala hospital, eastern Sudan. BMC Res Notes. 2011; 4(1): 311. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl-Moselhy EA, Khalifa HO, Amer SM, et al.: Risk factors and impacts of pre-Eclampsia: an epidemiological study among pregnant mothers in Cairo, Egypt. Journal of American Science. 2011; 7(5): 311–323. Reference Source\n\nBilano VL, Ota E, Ganchimeg T, et al.: Risk factors of pre-eclampsia/eclampsia and its adverse outcomes in low- and middle-income countries: a WHO secondary analysis. PLoS One. 2014; 9(3): e91198. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "65550",
"date": "06 Jul 2020",
"name": "Ishag Adam",
"expertise": [
"Reviewer Expertise Maternal and perinatal epidemiology"
],
"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 investigated an important topic “preeclampsia”. I have some points that need to be addressed.\nAbstract:\nNo need to have details of statistic methods in the abstract.\n\nMethods:\nIt is not clear if these cases were mild or severe cases or if these were early or late preeclampsia.\n\nMore elaborations are needed because the risk factors are different in the different types of preeclampsia.\n\nThere many factors that were not investigated e.g. anemia, malaria, body mass index, blood groups.\n\nI think all this information could be added to your work and you might have different results.\n\nSample size:\nIt is advisable to consider the difference between the two groups using the least prevalent one e.g. smoking or alcohol such as 3. 0 vs 2. % and not using the high one as you used antenatal care.\n\nStatistics:\nI do not think you used mix model I think you used logistic regression models.\n\nIn the methods, you mentioned that variables with p ≤ 0.2 were entered in the model. Then later on you contradict yourself and you mentioned that variables that were shown to be associated with preeclampsia were entered in the model regardless of their P value.\n\nResults:\nAll your tables can be summarized in two tables only: Table 1 all comparing all variables between preeclampsia and controls Table 2 logistic regression with non -adjusted and adjusted values\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "96070",
"date": "19 Oct 2021",
"name": "Jussara Mayrink",
"expertise": [
"Reviewer Expertise preeclampsia",
"high risk pregnancy",
"maternal mortality",
"near miss"
],
"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\nDear authors, there are some suggestions I have about your study:\n\nAbstract:\n\nBe careful with preeclampsia concept, which is not exclusively linked to proteinuria anymore. Methods section can be more concise and in the sentence “(…) multivariable mixed-effect logistic regression analysis using the significant variables from bivariable analysis (…)” I am not sure about the significant variables. Where are they?\n\nIntroduction:\n\nI missed the justification for the study. Why is it important to describe preeclampsia population from Nairobi, compared to the non-exposed group? In my opinion, authors should establish a link with preeclampsia prophylaxis, addressing social context and impossibility to perform laboratory markers to screen high-risk population.\n\nMethods:\nCase definition and recruitment: Since 2014 we recommend a “new” and “broader” concept of preeclampsia, which goes beyond the combination of hypertension and proteinuria. Thus, please, try to use other references. As this is a case-control study, it would be interesting adding some information, as the onset of preeclampsia, for instance. Interesting information is the criteria to diagnose preeclampsia.\n\nStudy design: I am afraid this is not a case-control study. The researches have looked to the information about cases and not-cases of preeclampsia and eclampsia. And, at the end, you perform a comparison between them on regard some characteristics. This is not a case-control study.\n\nResults and discussion:\nDear authors, every time you put a result without statistical significance you must add this information. Another comment is: There are too many tables!\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": []
}
] | 1
|
https://f1000research.com/articles/9-192
|
https://f1000research.com/articles/8-1744/v1
|
11 Oct 19
|
{
"type": "Research Article",
"title": "Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study",
"authors": [
"Endang W. Bachtiar",
"Atikah Cyntia Putri",
"Boy M. Bachtiar",
"Endang W. Bachtiar",
"Atikah Cyntia Putri"
],
"abstract": "Background: Salivary nitric oxide plays an important role as an antibacterial agent in the oral cavity. Here, we analyze salivary nitric oxide, Simplified Oral Hygiene Index (OHI-S) scores and the salivary flow rate in smokers and non-smokers which has not been done previously. Methods: A cross sectional study included 25 smokers and 25 non-smokers. Their OHI-S results were categorized as “good,” “medium,” or “bad.” Unstimulated saliva samples were collected, and their nitric oxide concentration was measured using the Griess method. Results: The salivary flow rate in smokers was lower, at 0.30 ml/minute, compared to non-smokers who had a salivary flow rate of 0.33 ml/minute. This was statistically insignificant. There was a significant difference in the concentrations of nitric oxide between smokers and non-smokers (p < 0.05). Smokers had higher concentrations than non-smokers (185.4 µM Vs 114.60 µM). In addition, there was a moderate positive correlation (r = 0.305) between the concentration of salivary nitric oxide level and the OHI-S results. Conclusions: It was concluded that salivary nitric oxide concentration was higher in smokers, and the oral hygiene condition of smokers was poor.",
"keywords": [
"Salivary Nitric Oxide",
"Oral Hygiene Index",
"Salivary Flow Rate",
"Smoker",
"Griess Method",
"Whole Saliva"
],
"content": "Introduction\n\nSalivary nitric oxide (NO) is a free radical that plays an important role as a regulator for various physiological and pathological body mechanisms. It can act as a nonspecific antimicrobial defense system in the oral cavity, either through inhibiting bacterial growth or enhancing macrophage-mediated cytotoxicity1,2. There are two main sources of salivary NO in the oral cavity; chemically from physiological reduction by the enzyme nitrate reductase, released by certain bacteria, that converts nitrate (NO3-) to nitrite (NO2-). The other is through a reduction process facilitated by certain bacterial products resulting from the dental plaque microflora, forming nitrogen oxide and nitric acid. Unstable and spontaneous nitric acid decomposes into NO3. NO can also be produced enzymatically from L-arginine, induced by nitric oxide synthase (NOS), an enzyme in the salivary gland2. NOS has three isoforms: neuronal NOS (nNOS; Type I, NOS-1); inducible NOS (iNOS; Type II, NOS-2) present in various cells and tissues, produced in immunocompetent cells, like macrophages, infected by bacteria involved in regulating inflammatory reactions; and endothelial NOS (eNOS; Type III, NOS-3) found in the endothelial vascular cells3,4. High levels of NO can cause inflammation, immune disorders, neurological diseases, atherosclerosis, and cancer4.\n\nThe relationship between oral health and the body's ability to produce nitric oxide is mainly part of a system known as an enterosalivary sequence. Nitric oxide (NO) levels in the oral cavity and saliva have been associated with various oral diseases. There is a hypothesis that the harmful effects of tobacco smoke may be the result of the accumulation of oxidative endothelial cell damage. Due to the presence of NO in tobacco smoke (about 500 ppm of NO and other active nitrogen oxides), it was expected that as a result of smoking, the NO metabolites in systemic circulation will increase5. Since smoking may affect the oral microenvironment, in this study the analysis was performed in relation to smoking habits.\n\n\nMethods\n\nThe study groups included 25 smokers and 25 non-smokers, aged 18–24 years, studying at the Universitas Indonesia; consecutive sampling was used from August to October 2018. A sample size of convenience was used. Invitation to participate was posted on announcement boards of the Faculty of Dentistry, Universitas Indonesia, to which participants responded via mobile telephone. This cross-sectional study obtained ethical approval from the ethical research committee of the Faculty of Dentistry, Universitas Indonesia, 2018 (89/Ethical Approval/FKGUI/VII/2018). Before samples were collected, written consent (informed consent) was obtained from participants according to the guidance provided by the Ethics Committee of Faculty of Dentistry, Universitas Indonesia.\n\nAll participants: 18–24 years old (late adolescence/young adulthood), in good health, absence of systemic disease or history of systemic disease, normal growth and development, not having consumed food for the two hours before sampling. We assessed young adults assuming that they had not suffered any age related disease that could interfere with levels of NO. Smokers: not having smoked in the hour prior to sampling, been smoking for more than six months and consume at least one tobacco cigarette per day (containing no cloves). Non-smokers: never smoked or not smoked for more than 5 years.\n\nAll participants: age <18 years or >24 years, having systemic or congenital diseases related to the oral cavity, currently undergoing medical therapy, currently taking local or systemic medication that might affect saliva composition.\n\nAll participants were interviewed in order to obtain all data, which were recorded using a data form (available as Extended data)6 prior to data entry into a computer spreadsheet. Each interview was conducted by two authors of this study team (ACP and EWB) at the Faculty of Health Science Universitas Indonesia. Depok Indonesia. Participants were instructed not to ingest food for a minimum of two hours before their saliva samples were collected. The smokers were classified into “light,” “moderate,” and “heavy” smokers based on the number of cigarettes consumed in a day (light:1-10 cigarettes, moderate; 11-20 cigarettes and heavy smoker, smoking more than 20 cigarettes), Individuals who had not smoked in the past 5 years were classified as non-smokers. According the study at the Behavioral Endocrinology Laboratory at Penn State University. Newport, one cigarette contains on average 13.4 mg nicotine. Hence we categorized in a day light smoker have consumed less than 134 mg nicotine, moderate smoker between 134-268 mg nicotine and heavy smoker more than 268 mg nicotine.\n\nOral hygiene was determined using the Simplified Oral Hygiene Index (OHI-S), comprising two components: plaque (debris) and calculus. The OHI-S was calculated on six tooth surfaces consisting of buccal surfaces in teeth 11, 16, 26, and 31; and lingual surfaces in teeth 36 and 467. The OHI-S categories and their corresponding scores were as follows, good (0.0-1.2) moderate (1.3-3.0) and bad (3.1-6.0)7.\n\nIn this study, the saliva samples collected were unstimulated whole saliva (UWS). Over a period of three minutes, all saliva was collected in a 14-mL Falcon tube and the resulting volume was divided to 3 to get the salivary flow rate per minute. Following this, a further sample was collected by having individual participants spit 1 ml saliva into a sterile 14 ml tube. It was then transferred and stored in 1.5 ml microcentrifuge tube containing 500 µL phosphate buffer saline (PBS) (Sigma-Aldrich Dorset UK. Cat.P4417) and a protease inhibitor 0.1M Phenyl methane sulfonyl fluoride (PMSF) (Sigma-Aldrich, Cat. 329-98-6). The saliva samples were centrifuged at 5000×g for 5 min at 4°C. This was done, to separate the supernatant and the pellet in the saliva. The supernatant, containing organic components, was then stored at -20ºC and the pellets were discarded. The NO concentration of the supernatant was determined by nitrite levels, using the Griess reaction (Sigma-Aldrich, Dorset, UK, Cat. G4410). Briefly, a duplicate of 50 µL of the supernatant and 50 µL of Griess reagents were mixed and incubated in the dark for 10 minutes at room temperature, then the mixture was read at 450 nm by a spectrophotometer (AccuReader. M965/M965+) Nangang, Taipei, Taiwan.\n\nData were analyzed using SPSS version 22.0. The Mann-Whitney U-test was used to analyze differences between smokers and non-smokers, ANOVA was used to analyze the significant difference between four groups (non-smokers, light smokers, moderate smokers, and heavy smokers). Spearman’s correlation was used to analyze the relationship between salivary flow rate or salivary nitric oxide concentration and OHI-S (for smokers only).\n\n\nResults\n\nThe results, after measuring the unstimulated salivary flow rate in 25 smokers and 25 non-smokers, showed that the flow rate in smokers was lower at 0.30 ml/minute, with a minimum value of 0.10 ml/minute and a maximum value of 1.67 ml/minute. In comparison, non-smokers had a flow rate of 0.33 ml/minute, with a minimum value of 0.06 ml/minute and a maximum value of 1 ml/minute (Figure 1). Analysis through the Normality test showed that the distribution of data was abnormal. There was no significant difference between unstimulated salivary flow rates in smokers and non-smokers according to the Mann-Whitney statistical test (p = 0.748).\n\nSFR, salivary flow rate.\n\nThere was a trend of decreasing salivary flow rate with increasing levels of smoking from “light,” to “moderate,” to “heavy”. The results of the Kruskal Wallis statistical test showed that the average unstimulated salivary flow rate in heavy smokers was lower than that of light and moderate smokers, with no significant difference between the smokers (p = 0.151). As shown in Figure 2, light smokers had an unstimulated salivary flow rate of 0.50 ml/minute (minimum value of 0.13 ml/minute and a maximum of 1.67 ml/minute). The unstimulated salivary flow rate in moderate smokers was 0.2830 ml/minute (minimum 0.10 ml/minute, and maximum value was 0.67 ml/minute). Heavy smokers had a salivary flow rate of 0.20 ml/minute with a minimum value of 0.10 ml/minute and a maximum value of 0.30 ml/minute.\n\nSFR, salivary flow rate.\n\nBased on the results of the Mann-Whitney statistical test, there was a significant difference between the concentrations of NO in smokers and non-smokers (p = 0.040). Smokers displayed higher salivary NO concentrations (185.4 µM; 114.60-295.10 µM) than non-smokers (139.5 µM; 97.70-538.4 µM) (Figure 3).\n\nAmongst the 25 smokers, 14 (56%) were light smokers, 8 (36%) were moderate smokers, and 3 (12%) were heavy smokers. Figure 4 shows an increasing trend in NO concentrations in the saliva with increased extent of smoking. ANOVA analysis found no significant difference in NO concentration (p = 0.600) between the different smoker groups. The concentration of NO in heavy smokers was higher than that for moderate and light smokers; 220.333 µM for heavy smokers, 196.950 µM for moderate smokers, and 183.642 µM for light smokers.\n\nRelationship between unstimulated whole salivary flow rate and the status of oral hygiene indicated by the OHI-S for the smoker group.\n\nResults of the Spearman’s correlation test to determine the relationship between salivary flow rate of the smokers and the status of oral and dental hygiene indicated by OHI-S scores, found an insignificant negative relationship (r = -0.118, p = 0.573). This is depicted in Figure 5.\n\nUWS, unstimulated whole saliva.\n\nThe results of the analysis of the relationship between NO concentrations and oral hygiene status assessed through the OHI-S of smokers are illustrated in Figure 6. The Spearman’s correlation test showed that there was a moderate positive but insignificant relationship (r = 0.305, p = 0.138) between the concentration of NO and the OHI-S.\n\n\nDiscussion\n\nNO is a key element in saliva that regulates various physiological and pathological mechanisms of the body and can act as an antibacterial agent2,4. Saliva can act as a biomarker for the occurrence of health problems, especially in the oral cavity, which correlates to responses from the immune system8. Using unstimulated saliva is an accurate method for analyzing salivary gland status through the salivary flow secreted per minute9.\n\nIn this study, we observed smoking habits can affect NO concentration in saliva. This was further associated with dental and oral hygiene. As a result of exposure of the oral cavity to the components of cigarettes, smoking habits can affect elements in the oral cavity, one of them being the salivary flow rate10. This study showed that the salivary flow rate for smokers was lower, compared to non-smokers, though the difference was not significant. The results of this study correlate with the results of Khan et al. which also stated the lack of a significant difference between salivary flow rates of smokers and non-smokers10. According to Singh et al. and Khan et al., there is a decrease in the flow rate of saliva in smokers compared to non-smokers owing to the heat and effect of toxins from cigarettes produced during smoking. There is a declined blood flow in the salivary glands, causing decreased flow of saliva from the disturbed salivary gland9,10.\n\nThe process of salivary secretion is quite complex, and the flow and composition of saliva can be influenced by several factors11. These include the duration and extent of smoking per day, the more cigarettes consumed and longer the duration of the oral cavity’s exposure to the content of cigarettes, the lower the flow of saliva in the mouth12,13. Previous studies reported that heavy smokers who indulged in regular smoking habits for more than 6 months, showed decreased salivary secretions compared to non-smokers9,13. In a study conducted by Fenoll-Palomares et al., there was an increase in salivary flow rate in smokers who smoked a short period, but the difference between salivary flow rates in long-term smokers and non-smokers was unknown14. In this study, as with the previous studies, the salivary flow rate of heavy smokers was lower than light and moderate smokers, indicating that smoking intensity affects the secretion of salivary flow rates. Data from Figure 2 indicates that light smokers can experience increased salivary gland activity leading to an increase in the rate of saliva flow, while those who have smoked for a longer period of time decrease the rate of saliva flow12.\n\nThe reduced flow rate of saliva can affect its protective function in the oral cavity. The content of cigarettes, can cause poor oral hygiene, halitosis, and lead to reddish and white local lesions15. Based on the results of this study, we found a weak negative relationship between salivary flow rates in smokers, and dental and oral hygiene conditions. Several studies have shown that oral hygiene conditions in patients who are smokers are worse in regard to dental plaque accumulation, compared to those who are non-smokers16,17. This is due to smoking habits that affect salivary flow rates as the parotid gland’s function of secreting dilute saliva, is adversely affected when exposed to cigarette smoke. This causes a change in the nature of the saliva secreted. For instance, the sublingual and submandibular glands will secrete mucin saliva, having a thicker consistency, increased salivary mucin can reduce other components of saliva such as immunoglobulins and enzymes, which have function as an inhibitor of plaque growth and so a reduced quantity of saliva can increase plaque formation18,19.\n\nChanges in salivary flow rate due to smoking can affect dental and oral hygiene. The physiological balance inside the oral cavity is disturbed due to smoking, demonstrated by the concentration of NO in the saliva20. This study found that NO concentrations in smokers were significantly higher than non-smokers. This, however, contrasts with the findings of Rezaei et al., where the concentration of NO in smokers was lower than that of non-smokers due to a decrease in the expression of the eNOS enzyme gene following exposure to cigarettes, causing decreased NO metabolism1. NO is produced from NO-forming enzymes, additional dietary or atmospheric NO intake, and the result of bacterial oxidation metabolism, which acts as a non-specific defense mechanism in the oral cavity to prevent excessive bacterial growth19,21. In this study, the increased concentration of NO was likely due to high bacterial activity, causing plaque, indicated by the high OHI-S.\n\nThe results of this study from the correlation tests conducted by the researchers reveal that there was a moderate positive but insignificant relationship between salivary NO concentration and the status of dental and oral hygiene, assessed using the OHI-S. This study is in line with the research conducted by Mobarak and Abdallah which showed no significant relationship between the concentration level of NO and the status of oral hygiene22. According to Bayindir et al., increased NO concentration correlated with poor oral and dental hygiene23. This is because the thickness of the plaque that is mature, inhibits the diffusion of oxygen into the deeper layers where the facultative anaerobic bacteria are located. Resultantly, the bacteria multiply owing to lack of oxygen, producing more NO because of increased bacterial activity.\n\nThe results of this study seem to indicate that in smokers if OHI-S increases it is followed by an increase in NO levels in saliva. This phenomenon may be due to an increase in plaque accumulation which might trigger NO production.\n\nThe potential bias in this study is the possibility of determining smoking frequency because it is only based on interviews. This research also has limitations in the number of samples because within the timeframe specified for recruiting participants only certain people are willing to participate as research subjects. The results of this study are expected to contribute to further research in order to find strategies to improve oral health in smokers.\n\n\nData availability\n\nOpen Science Framework: Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers. https://doi.org/10.17605/OSF.IO/WD2GV6.\n\nThis project contains the following underlying data:\n\nF1000 Raw data for NO concentrations (salivary NO concentrations of each participant).\n\nF1000 S-OHIS (raw data for plaque and calculus for each participant).\n\nOpen Science Framework: Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers. https://doi.org/10.17605/OSF.IO/WD2GV6.\n\nThis project contains the following extended data:\n\nQuesioner F1000 research (English translation of the questionnaire used in this study).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank Editage (www.editage.com) for their English language editing services.\n\n\nReferences\n\nRezaei F, Taghvai AM, Vaisi-Raygani A, et al.: A Comparative Study of Salivary Nitric Oxide Between Smokers and Nonsmokers. Sch Res Libr. 2016; 8(20): 145–50. Reference Source\n\nSurdilovic D, Stojanovic I, Apostolovic M, et al.: The Role of Nitric Oxide in Saliva in Reduction of Caries. 2008; 25(2): 93–5.\n\nWadhwa D, Bey A, Hasija M, et al.: Determination of levels of nitric oxide in smoker and nonsmoker patients with chronic periodontitis. J Periodontal Implant Sci. 2013; 43(5): 215–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPreethi S, Jose JI, Sivapathasundharam B, et al.: Evaluation of Salivary Nitric Oxide Levels in Smokers, Tobacco Chewers and Patients with Oral Lichenoid Reactions. J Clin Diagnostic Res. 2016; 10(1): ZC63–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNene S, Gelabert H, Moore W, et al.: Cigarette smoking increases endothelial-derived vasorelaxation in the rat carotid artery in a dose-dependent manner. J Surg Res. 1997; 71(2): 101–6. PubMed Abstract | Publisher Full Text\n\nBachtiar EW: Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers. 2019. http://www.doi.org/10.17605/OSF.IO/WD2GV\n\nSimplified Oral Hygiene Index | OHI-S. 2010. Reference Source\n\nRezaei F, Taghvai AM, Vaisi-raygani A, et al.: A Comparative Study of Salivary Nitric Oxide Between Smokers and Nonsmokers. Sch Res Libr. 2016; 8(20): 145–50. Reference Source\n\nChakrabarty S, Patil S, Bandalore SRH, et al.: Long-term effect of tobacco on resting whole mouth salivary flow rate and pH: An institutional based comparative study. J Indian Acad Oral Med Radiol. 2015; 27(4): 549–52.\n\nSingh M, Yadav P, Ingle N, et al.: Effect of long-term smoking on salivary flow rate and salivary pH. J Indian Assoc Public Heal Dent. 2015; 13(1): 11–13. Publisher Full Text\n\nKhan GJ, Javed M, Ishaq M: Effect of Smoking on Salivary Flow Rate. Gomal J Med Sci. 2010; 8(2): 221–4. Reference Source\n\nAlaee A, Azizi A, Valaei N, et al.: The Correlation Between Cigarette Smoking and Salivary Flow Rate. J Res Dent Maxillofac Sci. 2017; 2(3): 5–9. Reference Source\n\nRad M, Kakoie S, Niliye Brojeni F, et al.: Effect of Long-term Smoking on Whole-mouth Salivary Flow Rate and Oral Health. J Dent Res Dent Clin Dent Prospects. 2010; 4(4): 110–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFenoll-Palomares C, Muñoz-Montagud JV, Sanchiz V, et al.: Unstimulated salivary flow rate, pH and buffer capacity of saliva in healthy volunteers. Rev Esp Enferm Dig. 2004; 96(11): 773–83. PubMed Abstract | Publisher Full Text\n\nRehan F, Khan RB, Khurshid Z, et al.: Analysis of Resting Mouth Salivary Flow Rate and Salivary pH of Tobacco Chewers and Smokers. JPDA J Pak Dent Assoc. 2016; 25(254): 158–63. Reference Source\n\nPereiraa ADFV, Castrob ACS, et al.: Effects of Cigarette Smoking on Oral Hygiene Status. J Dent Sci. 2013; 28(1): 4–7. Reference Source\n\nOlusegun S, Ayanbadejo P, Savage KW, et al.: Oral Hygiene Status and Periodontal Treatment Needs of Nigerian Male Smokers. TAF Prev Med Bull. 2010; 9(2): 107–12. Reference Source\n\nAlfianur N, Suryana B: Pengaruh Viskositas Saliva Terhadap Pembentukan Plak Gigi. Insidental. 2014; 1(1).\n\nPetrušić N, Posavac M, Sabol I, et al.: The Effect of Tobacco Smoking on Salivation. Acta Stomatol Croat. 2015; 49(4): 309–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBodis S, Haregewoin A: Evidence for the release and possible neural regulation of nitric oxide in human saliva. Biochem Biophys Res Commun. 1993; 194(1): 347–50. PubMed Abstract | Publisher Full Text\n\nHegde MN, Kumari S, Hegde N: Evaluation of The Status of Salivary Nitric Oxide in Patients with Dental Caries. J Heal Sci. 2012; 2(2): 6–9. Reference Source\n\nMobarak EH, Abdallah DM: Saliva Nitric Oxide Levels in Relation to Caries Experience and Oral Hygiene. J Adv Res. 2011; 2(4): 357–62. Publisher Full Text\n\nBayindir YZ, Polat MF, Seven N: Nitric oxide concentrations in saliva and dental plaque in relation to caries experience and oral hygiene. Caries Res. 2005; 39(2): 130–3. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "60176",
"date": "03 Mar 2020",
"name": "Juni Handajani",
"expertise": [
"Reviewer Expertise Oral Biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study analyzed Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study. The results of the study explained the relationship between unstimulated whole salivary flow rate and the status of oral hygiene also the relationship between salivary nitric oxide concentration and the status of oral hygiene assessed through the OHI-S of smokers but the conclusions do not explain the correlation as mentioned.\nThis paper is good and is an important addition to the literature. The concept of correlation between salivary nitric oxide and the status of oral hygiene index is written clearly. I would like to give the suggestion of adding explanations about why the authors correlated flow rate saliva and status of oral hygiene or add literature that explains about the effect oral hygiene status on salivary flow rate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5293",
"date": "16 Mar 2020",
"name": "Boy Muchlis Bachtiar",
"role": "Author Response",
"response": "Dear Reviewer I am pleased to resubmit for publication the revised version of Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study We appreciate all the insightful comments provided by the reviewer. Based on the reviewer’s guidance, includes a number of positive changes, we endeavored to improve the fit of the paper with the journal. We hope that these revisions improve the paper such that Editor and the Reviewer now deem it worthy of publication in F1000 Research. Thank you for taking the time to help us improve the paper. Sincerely, Authors : 1. Endang W Bachtiar 2. Atikah Cyntia Putri 3. Boy M Bachtiar Author’s response to reviewer comments Comment provided by Reviewer #1 This study analyzed Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study. The results of the study explained the relationship between unstimulated whole salivary flow rate and the status of oral hygiene also the relationship between salivary nitric oxide concentration and the status of oral hygiene assessed through the OHI-S of smokers but the conclusions do not explain the correlation as mentioned. This paper is good and is an important addition to the literature. The concept of correlation between salivary nitric oxide and the status of oral hygiene index is written clearly. I would like to give the suggestion of adding explanations about why the authors correlated flow rate saliva and status of oral hygiene or add literature that explains about the effect oral hygiene status on salivary flow rate. Author’s response: We thank the reviewer for the suggestions. As suggested by the reviewer, we have added sentence (line 193-200): Changes in salivary flow rate due to smoking can affect dental and oral hygiene.The study by Dawes and Watanabe reveals that reducing the stimulated salivary flow rate, which retards the rate of salivary clearance of sugar from the oral cavity.20Decreasing of the clearance of sugar might be followed by a decrease of OHIS.The increase of salivary flow is much more effective in neutralizing and buffering food acids. The study by Shimazaki et al. reveals that decreased saliva secretion affects both dental caries and general periodontal health status.21 Ahmad et al also reported that hyposalivation due to thelow saliva flow rate related to more caries in the aging population and significantly poor of the quality of life.22"
}
]
},
{
"id": "60322",
"date": "04 Mar 2020",
"name": "Muhammad S. Zafar",
"expertise": [
"Reviewer Expertise Dentistry",
"oral sciences"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have reviewed the manuscript “Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study” submitted to “F1000Research” for publication. I found this work interesting and fit well with in the scope of this journal. In this cross-sectional study, authors have analyzed and compared salivary nitric oxide, Simplified Oral Hygiene Index (OHI-S) scores and the salivary flow rate in smokers and non-smokers. The manuscript fits well within the scope of the journal; it needs some major improvements; there are a few suggestions that authors may consider to improve it further:\nAbstract: is structured and has been described clearly; however, there is no description of statistical analysis that should be added to the abstract.\n\nIntroduction; is brief however covering the background information and the rationale of the study effectively.\n\nMethodology and results have been reasonably presented; OHIS score should be defined.\n\nFigure 1 and 2: SFR legend is not required and must be deleted to match the style of figure 3.\n\nFigure 5 and 6: Use same style to describe OHIS; either OHIS or OHI-S?\n\nThe discussion is a little bit brief; authors must include and cite more studies in context comparing their findings with previous studies. For example; why specific saliva collection devices were not used and what are the possible limitations for using Falcon tubes? The authors could include the following paper with relevant information:\nKhurshid, Zohaib, et al. \"Human saliva collection devices for proteomics: An update.\" International journal of molecular sciences 17.6 (2016): 846.1\n\nSimilarly, how the reduced salivary flow and age may affect the OH and quality of life should be discussed; the information from the following papers could be included:\nAhmad, Mohammad S., et al. \"The impact of hyposalivation on quality of life (QoL) and oral health in the aging population of Al Madinah Al Munawarrah.\" International journal of environmental research and public health 14.4 (2017): 445.2\nKhurshid, Zohaib, et al. \"Human saliva and its role in oral & systemic health.\" JPDA 25.04 (2016): 171.\n\nWhat are the final conclusions of this study? The key outcome points should be highlighted\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5294",
"date": "16 Mar 2020",
"name": "Boy Muchlis Bachtiar",
"role": "Author Response",
"response": "Dear Reviewer,I am pleased to resubmit for publication the revised version of Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional studyWe appreciate all the insightful comments provided by the reviewer. Based on the reviewer’s guidance, includes a number of positive changes, we endeavored to improve the fit of the paper with the journal.We hope that these revisions improve the paper such that Editor and the Reviewer now deem it worthy of publication in F1000 Research. Thank you for taking the time to help us improve the paper. Sincerely,Authors : 1. Endang W Bachtiar 2. Atikah Cyntia Putri 3. Boy M BachtiarComment provided by Reviewer #2I have reviewed the manuscript “Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study” submitted to “F1000Research” for publication. I found this work interesting and fit well with in the scope of this journal. In this cross-sectional study, authors have analyzed and compared salivary nitric oxide, Simplified Oral Hygiene Index (OHI-S) scores and the salivary flow rate in smokers and non-smokers.The manuscript fits well within the scope of the journal; it needs some major improvements; there are a few suggestions that authors may consider to improve it further:Abstract: is structured and has been described clearly; however, there is no description of statistical analysis that should be added to the abstract. Introduction; is brief however covering the background information and the rationale of the study effectively. Methodology and results have been reasonably presented; OHIS score should be defined. Figure 1 and 2: SFR legend is not required and must be deleted to match the style of figure 3. Figure 5 and 6: Use same style to describe OHIS; either OHIS or OHI-S? The discussion is a little bit brief; authors must include and cite more studies in context comparing their findings with previous studies. For example; why specific saliva collection devices were not used and what are the possible limitations for using Falcon tubes? The authors could include the following paper with relevant information: Khurshid, Zohaib, et al. \"Human saliva collection devices for proteomics: An update.\" International journal of molecular sciences 17.6 (2016): 846.1 Similarly, how the reduced salivary flow and age may affect the OH and quality of life should be discussed; the information from the following papers could be included: Ahmad, Mohammad S., et al. \"The impact of hyposalivation on quality of life (QoL) and oral health in the aging population of Al Madinah Al Munawarrah.\" International journal of environmental research and public health 14.4 (2017): 445.2 Khurshid, Zohaib, et al. \"Human saliva and its role in oral & systemic health.\" JPDA 25.04 (2016): 171. What are the final conclusions of this study? The key outcome points should be highlighted Author’s response: Line 60: OHIS score has been defined. Figure 1 and 2: SFR legend has been deleted. Figure 5 and 6: we use OHIS. Discussion section has been revised according to the reviewer suggestion and the suggested references have been added. (Line 193-200, line 227-230). We have stated the final conclusion in Line 220-222"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1744
|
https://f1000research.com/articles/8-1740/v1
|
11 Oct 19
|
{
"type": "Research Article",
"title": "Prevalence of periodontal diseases and its correlation with different risk factors among an adult Egyptian population: a cross-sectional study",
"authors": [
"Marwa M.S. Abbass",
"Dina Rady",
"Israa Ahmed Radwan",
"Sara El Moshy",
"Nermeen AbuBakr",
"Mohamed Ramadan",
"Nermin Yussif",
"Ayoub Al-Jawaldeh",
"Dina Rady",
"Israa Ahmed Radwan",
"Sara El Moshy",
"Nermeen AbuBakr",
"Mohamed Ramadan",
"Nermin Yussif",
"Ayoub Al-Jawaldeh"
],
"abstract": "Background: Even though extensive studies on the prevalence of periodontal diseases in various populations worldwide have been carried out, data for the Egyptian population is limited. The present study was carried out to evaluate the prevalence and the severity of periodontal disease and its correlation with different risk factors. Methods: Periodontal examination was performed on 343 adults attending the outpatient clinics of the Faculty of Dentistry, Cairo University, as well as three private clinics. Socio-demographic data, brushing frequency, body mass index (BMI) and dietary habits were recorded using a questionnaire. Results: It was found that 58.9% of participants had calculus deposits. The prevalence of periodontitis was 89.8%, where 70.8% of participants had stage I and 15.2% had stage II, while only 4.4% and 2.05% suffered from stage III and stage IV, respectively. Calculus was positively correlated with age, grains, and sugar in drinks and negatively correlated with socioeconomic status, education level, brushing frequency and milk. Calculus was not correlated with gender and BMI. Periodontitis was positively correlated with age, carbohydrates other than bread, grains, crackers, and caffeinated drinks, while negatively correlated with gender, socioeconomic status, brushing frequency. Periodontitis was not correlated with BMI or education level. Conclusion: The present study clarifies that age, brushing frequency, carbohydrates and caffeinated drinks consumption are significant factors influencing the prevalence and the severity of periodontal diseases.",
"keywords": [
"calculus",
"periodontitis",
"prevalence",
"risk factors"
],
"content": "Introduction\n\nPeriodontitis can be defined as a chronic, progressive inflammatory disease affecting the periodontium surrounding the tooth. It eventually results in deterioration of the tooth-supporting apparatus and may result in tooth loss if untreated1.\n\nPeriodontal diseases, as well as dental caries, are considered the most widespread oral diseases worldwide2,3. It has been estimated that about 20–50% of the entire global population suffers from periodontal disease4. Residents of developing countries are more prone to periodontal diseases as compared to those of developed countries due to lack of awareness, lack of proper oral hygiene measures, a relatively expensive dental care system and lower socioeconomic status (SES)2.\n\nPeriodontal diseases have been linked to increased incidence of multiple systemic diseases such as cardiovascular diseases, metabolic diseases, possible complications of pregnancy, rheumatoid arthritis, respiratory diseases and kidney diseases5. Moreover, periodontal diseases have been also associated with increased risk of malignancies of the oral cavity as well as other sites6.\n\nIn 2014, the WHO reported a high prevalence of periodontal diseases in Egypt, 80% of the studied subjects suffered from periodontal diseases7. Despite the high prevalence of periodontal diseases in the Egyptian population, no definite preventive measures are undertaken to screen, prevent or to address this important health issue. Moreover, there is no precedent work correlating the prevalence of periodontal diseases with risk factors including dietary habits in the Egyptian population. Therefore, the aim of the present study is to investigate this amongst the Egyptian population.\n\n\nMethods\n\nThis study was carried out according to the regulations of the Ethics Committee, Faculty of Dentistry, Cairo University, Egypt (approval: 171217). Convenience sample was utilized in this study. Eligible patients were recruited according to the inclusion and exclusion criteria over a period of two months, starting from the 16th of August 2018 until the 18th of October 2018. Patients were recruited from the outpatient clinics at the Faculty of Dentistry, Cairo University, as well as three private dental offices (Cairo Dental Clinic, Specialized Dental Clinic and El-Rahamn Medical Center). Patients were asked directly to participate in the study while they were attending the clinics. Written consent was obtained from the patients to perform the examinations and for the use and publication of their anonymized data. The inclusion criteria were as follows: age: 18–74 years; gender: males and females; ethnicity: Egyptians. Exclusion criteria were smokers; previous history of/current radiotherapy and/or chemotherapy; pregnant or lactating females; edentulous patients; patients undergoing orthodontic therapy; patients with aggressive periodontitis; patients who had undergone periodontal treatment (including prophylaxis) and/or antibiotic therapy over the past three months.\n\nAccording to the following simple formula8: n'=NZ2P(1–P)d2(N–1)+Z3P(1–P)\n\nWhere n' = sample size with finite population correction, N = population size, Z = Z statistic for a level of confidence which is conventional (Z value is 1.96), p = expected prevalence and d = precision (5%, d = 0.05). The sample size was estimated to be 339 as the population of Egypt was considered to be 90,000,000, as estimated by the World Bank. The prevalence was estimated to be 32% by averaging the prevalence in India and Bangladesh of 17.5 –21.4%9 and 45% in India10.\n\nData were collected using a questionnaire that has previously been used in other studies11,12 with questions on age, sex, occupation, address, level of education and dietary habits (provided as Extended data)13. The questionnaire was filled out by the examiners in the clinics. A Beurer scale (Ulm, Germany) was used to measure body weights with individuals wearing clothing but no shoes. Standing heights were obtained. Body mass index (BMI) was calculated from measured height and weight data. Subjects were classified into the following groups: underweight (BMI<18.5 kg/m2); normal weight (BMI 18.5–24.9 kg/m2); overweight (BMI 25.0–29.9 kg/m2); obese (BMI ≥30.0 kg/ m2). Moreover, patients were categorized into low, moderate and high socioeconomic subgroups based on their education level, occupation, address and the health center where they received their treatments according to a validated socioeconomic status scale for health research in Egypt14.\n\nClinical and radiographic case identification was performed by trained examiners (MM and NY) to reach a consensus according to the latest classification of periodontal diseases that was described in 201815,16. The clinical outcomes were the assessment of the presence or absence of calculus and the stage of periodontitis. In order to define the stage of periodontitis, pocket depth (PD) and clinical attachment level (CAL) were measured using a Williams periodontal probe. Periodontitis was categorized into four stages (Table 1)16.\n\nCAL, clinical attachment loss.\n\nData were statistically described in terms of number of cases and percentages. Comparison between the study groups was done using ANOVA test with post-hoc multiple two-group comparisons. For comparing categorical data, Chi-square (χ2) test was performed. Correlation between variables was done using Spearman rank correlation equation. p values <0.05 were considered statistically significant. All statistical calculations were done using IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.\n\n\nResults\n\nThe number of individuals at each stage of the study are shown in Figure 1 and the number and percentage of patients in different categories are presented in Table 2. It was found that 24.5% of participants brush their teeth twice daily, while 23.3% don't brush their teeth17. The prevalence of calculus was 58.9%. The prevalence of periodontitis was 89.8%, where 70.8.5% of participants had stage I periodontitis and 15.2% had stage II, while 4.4.% and 2.04% of participants had stage III and stage IV, respectively.\n\nAs shown in Table 3, the highest percentage of calculus among different age groups was recorded in adults aged 50–70 years (70%). A comparison of the prevalence of calculus between age subgroups revealed a statistically significant difference (p =0.001). There was a positive correlation between age and calculus (rho=-0.192, p <0.001).\n\nThe correlation coefficient, rho, ranges from -1 to +1, where 1 = perfect positive correlation, 0 = no correlation, -1 = perfect negative (inverse) correlation. *Statistical significance at p-value < 0.05.\n\nRegarding gender and BMI, males and obese adults had the highest prevalence of calculus (55.4% and 65.8%, respectively). A comparison of the prevalence of calculus between gender subgroups as well as a comparison between BMI subgroups were statistically insignificant (p ≥0.05). There was no correlation between either of these factors and calculus (rho=-0.086, p=0.111 and rho=-0.101, p =0.062, respectively).\n\nRegarding SES, education level and brushing frequency, adults with a low SES, a low educational level and those who don’t brush their teeth had the highest prevalence of calculus (72.3%, 80% and 78.8%, respectively). A comparison of calculus prevalence between SES, education level and brushing frequency subgroups revealed a statistically significant difference (p=<0.05) and there was an inverse correlation between these factors and calculus (rho=-0.254, p<0.001; rho=-0.167, p=0.002; and rho=-0.326, p <0.001, respectively).\n\nAdults who consume bread, carbohydrates other than bread, eggs, fruits and vegetables, milk, milk products, candies and citrus juices less than or equal to two times a week had the highest prevalence of calculus compared to those who consumed these products more frequently (87.5%, 60.8%, 61.3%, 69.1%, 65%, 60.8%, 61.8% and 60.8%, respectively). Those who consume grains, sugars in drinks, sugar not in drinks, jams, crackers, junk food, chocolates, juices and caffeinated drinks with a frequency of one to six times per day had the highest prevalence of calculus (65.4%, 62.8%, 65.1%, 59%, 61.8%, 60.4%, 63.3%, 60.4% and 60.8%, respectively), as well as those who consume soda three to six times per week (62.8%).\n\nA comparison of calculus prevalence between consumption frequency subgroups for all dietary elements was statistically insignificant except for milk, grains and sugars in drinks (p<0.05). There was a positive correlation between consumption frequency of grain, sugars in drinks and calculus (rho=0.133, p=0.014 and rho=0.139, p=0.010, respectively), while milk revealed an inverse correlation (rho= -0.133, p =0.013).\n\nAs it is revealed in Table 3, the highest prevalence of periodontitis among different age groups (96.7%) was recorded among adults aged (50–70 years). In all age groups, the majority of participants suffered from stage I periodontitis: 70.5% of adults aged 18–34 years; 66.3% of adults aged 35–49 years; and 63.3% of adults aged 50–70 years. A comparison of periodontitis prevalence between age subgroups revealed a statistically significant difference (p=0.005). There was a positive correlation between age and periodontitis (rho=0.206, p=<0.001).\n\nThe highest percentage of periodontitis was recorded among males (92.1%), while in females the prevalence was 88.2 %. Stage I periodontitis was predominant, with 64.7% of males and 70.6% of females with this stage of periodontitis. A comparison of periodontitis prevalence between gender subgroups showed a statistically insignificant difference (p=0.115). There was a correlation between male gender and periodontitis (rho= -0.129, p=0.017).\n\nAmong different BMI groups, the highest prevalence of periodontitis was among obese participants (93.7%). Stage I periodontitis was the predominate stage, with 100% of underweight, 69.3% of normal, 63.7% of overweight adults and 73.4% of obese participants in this stage of the disease. A comparison of periodontitis prevalence between BMI subgroups revealed a statistically insignificant difference (p≥0.05). There was no correlation between BMI and periodontitis (rho=0.081, p=0.137).\n\nRegarding SES and education levels, participants with a low SES and a low educational level had the highest prevalence of periodontitis (94.7% and 98%, respectively). In all SES and education level subgroups, most participants had stage I periodontitis (Table 3). A comparison of periodontitis between SES subgroups revealed a statistically insignificant difference (p ≥0.05) while there was a statistically significant difference (p =0.001) between education level subgroups. There was no correlation between periodontitis and education level (rho= -0.009, p=0.067), while an inverse correlation was found between periodontitis and SES (rho=-0.176, p =0.001).\n\nIn the present study, all adults who reported that they don’t brush their teeth had periodontitis (100%). The majority of participants in all brushing frequency subgroups suffered from stage I periodontitis (Table 3). A comparison of periodontitis prevalence between subgroups revealed a statistically significant difference (p=0.003). There was an inverse correlation between brushing frequency and periodontitis (rho= -0.234, p = <0.001).\n\nA comparison of periodontitis prevalence between consumption frequency subgroups for all dietary elements was statistically insignificant except for the consumption of other carbohydrates, eggs, grains, soda and citrus juices (p>0.05). The consumption frequencies of carbohydrates other than bread, grains, crackers and caffeinated drinks were shown to have a positive correlation with periodontitis (rho=0.142, p=0.008; rho=0.181, p=0.001; rho=0.111, p=0.04; and rho=0.114, p=0.034, respectively). Moreover, the consumption frequencies of sugar in drinks and candies were very close to a significant positive correlation with periodontitis (rho=0.105, p=0.053 and rho=0.105, p=0.052, respectively). For the consumption of all foods at all frequencies, the majority of participants suffered from stage I periodontitis (Table 3).\n\n\nDiscussion\n\nSurveying the prevalence of periodontal diseases is challenging because of case misclassification and the number of teeth and sites to be examined18. According to the Canadian Health Measures Survey, the measurement of periodontal ligament attachment loss is the gold standard in reporting the prevalence of periodontal disease19.\n\nIn the current study, a new classification was utilized, where periodontitis is graded into stages according to the severity as well as the complexity of the treatment required to eliminate local risk factors. This classification is advantageous over others as it gives an idea about the severity, diagnosis, pathogenesis and the required treatment of periodontal conditions15,16.\n\nIn this study, there were positive correlations found between calculus, periodontitis, and age. It is well established that periodontal destruction is associated with periodontal disease activity, which is cumulative and tends to increase with age20.\n\nMale gender was correlated with the severity of periodontitis in the present investigation. Similar findings have been reported in a previous study conducted in southern Thailand21. This could be attributed to neglected oral-hygiene measures in males. Moreover, sex differences in periodontal disease may be due to gender-based heterogeneity in immune responses22.\n\nA negative correlation was found in the current study between periodontal health and SES, as well as a negative correlation was detected between the level of education and calculus among the studied participants. Other authors concur SES23,24, and education23,25, among other factors, that are influential on oral and periodontal health. Patients with low SES usually lack proper dental education, fail to visit the dentist on a regular basis and usually seek the dentist only in case of symptomatic complains24. The level of individual education is a component of SES. Individuals with higher education levels usually have a higher income and higher SES and are more likely to have routine, prophylactic dentist visits26. Moreover, education level influences the patient’s oral hygiene practice and dietary habits27. These factors and their associated psychological stresses negatively impact oral health through increasing inflammatory mediators and stimulating inflammation and altering host immune response to bacterial insult28.\n\nAnother risk factor for periodontal disease is poor oral hygiene, associated with the accumulation of plaque and calculus that result in gingivitis, which eventually results in periodontitis if untreated29. This is in accordance with the findings of the current study, which revealed a negative correlation between the frequency of teeth brushing and the presence of calculus and periodontitis.\n\nAlthough the influence of dietary habits on dental caries is more significant as compared to their influence on periodontal disease; nonetheless, a poor diet can negatively affect periodontal tissues, causing rapid progression of periodontal disease30. Malnutrition can modulate the inflammatory process and immune response28, which subsequently may cause periodontal disease31. One proposed mechanism through which nutrition can influence periodontal health is reactive oxygen species (ROS) and oxidative stresses. The presence of excessive oxidants can result in tissue damage via oxidation of important molecules, production of pro-inflammatory mediators as well as local and systematic inflammation, which negatively affects periodontal health31.\n\nMany dietary components, such as fats and sugars, can cause oxidative stress and increased ROS production, which promotes inflammatory processes32 and negatively impacts periodontal health33. Additionally, a sugary diet is linked to increased plaque formation. This could explain the positive correlation observed in the current study between sugar in drinks and calculus deposits and between intake of carbohydrates other than bread and crackers and periodontitis. Similar linkage between a high sugary diet and increased risk of periodontal disease and calculus deposits have been reported in previous studies33.\n\nIn the present work, a negative correlation between calculus and milk consumption has been reported. These results support the findings of Adegboye et al., who reported that dairy calcium, particularly from milk, is associated with a reduced risk of periodontitis34.\n\nHeavy coffee consumption was linked to an increased risk of periodontitis in the Korean population35. Likewise, a positive correlation was detected between the consumption of caffeinated drinks and periodontitis in the current study. This can be ascribed to their sugar as well as their caffeine content. Caffeine has been reported to increase alveolar bone loss in rats with induced periodontitis and reduce bone healing following teeth extraction36. Caffeine can enhance osteoclastic activity and suppress osteoblasts proliferation37. On the contrary, Machida et al.38 reported an inverse association between coffee consumption and periodontitis. The discrepancy in the reported effect of coffee on the alveolar bone can be attributed to different dosages of coffee and caffeine administered in each experiment.\n\nA healthy diet rich in fibers and whole grains intake is associated with reduced risk of periodontitis in several populations39,40. This is owing to the health benefits of whole grain, as they are rich in antioxidants and fibres41. Antioxidant intake has been positively associated with periodontal health30,42.\n\nIn the current study, a positive association between grain intake and periodontitis was observed. According to Hassan-Wassef43, the most commonly consumed grain in Egypt is fava beans. In the Egyptian cuisine, dried fava beans are slowly stewed overnight before being served. Therefore, it could be deduced that the boiling of beans has a negative impact on its antioxidant content44. Moreover, they are usually served alongside bread and combined with unsaturated fats and oils and many Egyptians consume fava beans from street vendors, which could be above-mentioned factors may alter host inflammatory response and negatively impact oral and periodontal health.\n\nEven though the current work investigated the prevalence of periodontal diseases in correlation to different risk factors, important risk factors still need to be investigated such as smoking and glycosylated haemoglobin level.\n\nIn conclusion, periodontitis is a multifactorial disease with many risk factors. Its progression is dependent on the interaction between intricate parameters, which pave the way to bacteria-induced inflammation and tissue destruction. A proper oral hygiene regime and nutrient-rich healthy diet in addition to prophylactic dental visits can reduce the risk of periodontal diseases and promote oral health.\n\n\nData availability\n\nFigshare: Raw data for periodontitis 2.xlsx. https://doi.org/10.6084/m9.figshare.9756428.v117\n\nFigshare: questionnaire periodontitis adult.docx. https://doi.org/10.6084/m9.figshare.9756542.v113\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\nAuthors would like to acknowledge the support and technical guidance of nutrition unit at World Health Organization office for Eastern Mediterranean region.\n\n\nReferences\n\nNovak M: Classification of diseases and conditions affecting the periodontium. In: Newman. M, Takei. H, Carranza. F, eds. Clinical Periodontology. 9th ed. Philadelphia: Saunders Publishers an Imprint of Elsevier Science. 2003; 64–73.\n\nPage RC, Beck JD: Risk assessment for periodontal diseases. Int Dent J. 1997; 47(2): 61–87. PubMed Abstract | Publisher Full Text\n\nRaitapuro-Murray T, Molleson TI, Hughes FJ: The prevalence of periodontal disease in a Romano-British population c. 200-400 AD. Br Dent J. 2014; 217(8): 459–466. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanz M, D’Aiuto F, Deanfield J, et al.: European workshop in periodontal health and cardiovascular disease—scientific evidence on the association between periodontal and cardiovascular diseases: a review of the literature. Eur Hear J Suppl. 2010; 12(suppl_B): B3–B12. Publisher Full Text\n\nNazir MA: Prevalence of periodontal disease, its association with systemic diseases and prevention. Int J Health Sci (Qassim). 2017; 11(2): 72–80. PubMed Abstract | Free Full Text\n\nMichaud DS, Fu Z, Shi J, et al.: Periodontal Disease, Tooth Loss, and Cancer Risk. Epidemiol Rev. 2017; 39(1): 49–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nResults of epidemiological study on oral health status. Reference Source\n\nMetcalfe C: Biostatistics: A Foundation for Analysis in the Health Sciences. 7th edn. Wayne W, Daniel, Wiley, 1999. No. of. pages: xiv+ 755+ appendices. ISBN 0‐471‐16386‐4 Stat Med. 2001; 20(2): 324–326.\n\nJacob PS: Periodontitis in India and Bangladesh. Need for a population based approach in epidemiological surveys. A Literature review. Bangladesh J Med Sci. 2010; 9(3): 124–130. Publisher Full Text\n\nNaseem S: Oral and Dental Diseases: Causes, Prevention and Treatment Strategies: Burden of Disease. 2005; 275-298. Reference Source\n\nAbbass MMS, Mahmoud SA, El Moshy S, et al.: The prevalence of dental caries among Egyptian children and adolescences and its association with age, socioeconomic status, dietary habits and other risk factors. A cross-sectional study [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Res. 2019; 8: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbbass MMS, AbuBakr N, Radwan IA, et al.: The potential impact of age, gender, body mass index, socioeconomic status and dietary habits on the prevalence of dental caries among Egyptian adults: a cross-sectional study [version 1; peer review: 3 approved]. F1000Res. 2019; 8: 243. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbbass M: questionnaire periodontitis adult.docx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9756542.v1\n\nEl-Gilany A, El-Wehady A, El-Wasify M: Updating and validation of the socioeconomic status scale for health research in Egypt/Mise à jour et validation d'une échelle du statut socioéconomique pour la recherche en santé en Égypte. East Mediterr Health J. 2012; 18(9): 962–968. PubMed Abstract\n\nTonetti MS, Greenwell H, Kornman KS: Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol. 2018; 89 Suppl 1: S159–S172. PubMed Abstract | Publisher Full Text\n\nPapapanou PN, Sanz M, Buduneli N, et al.: Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Clin Periodontol. 2018; 45 Suppl 20: S162–S170. PubMed Abstract | Publisher Full Text\n\nAbbass M: Raw data for periodontitis 2.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9756428.v1\n\nLocker D, Slade GD, Murray H: Epidemiology of periodontal disease among older adults: a review. Periodontol 2000. 1998; 16: 16–33. PubMed Abstract | Publisher Full Text\n\nHealth Canada: Report on the Findings of the Oral Health Component of the Canadian Health Measures Survey. 2007-2009. [Last accessed on 2014 Nov 07]. Reference Source\n\nDye BA: Global periodontal disease epidemiology. Periodontol 2000. 2012; 58(1): 10–25. PubMed Abstract | Publisher Full Text\n\nBælum V, Pisuithanakan S, Teanpaisan R, et al.: Periodontal conditions among adults in Southern Thailand. J Periodontal Res. 2003; 38(2): 156–163. PubMed Abstract | Publisher Full Text\n\nGrover V, Jain A, Kapoor A, et al.: The Gender Bender effect in Periodontal Immune Response. Endocr Metab Immune Disord Drug Targets. 2016; 16(1): 12–20. PubMed Abstract | Publisher Full Text\n\nAlmerich-Silla JM, Almiñana-Pastor PJ, Boronat-Catalá M, et al.: Socioeconomic factors and severity of periodontal disease in adults (35-44 years). A cross sectional study. J Clin Exp Dent. 2017; 9(8): e988–e994. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKadtane SS, Bhaskar DJ, Agali C, et al.: Periodontal Health Status of Different Socio-economic Groups in Out-Patient Department of TMDC & RC, Moradabad, India. J Clin Diagn Res. 2014; 8(7): ZC61–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonfim Mde LC, Mattos FF, Ferreira e Ferreira E, et al.: Social determinants of health and periodontal disease in Brazilian adults: a cross-sectional study. BMC Oral Health. 2013; 13: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatson CA, Nilam S: Educational Level as a Social Determinant of Health and Its Relationship to Periodontal Disease as a Health Outcome. J Dent Sci Ther. 2017; 1(3): 8–11. Publisher Full Text\n\nGomes AP, da Silva EG, Gonçalves SH, et al.: Relationship between patient ’s education level and knowledge on oral health preventive measures. Int Dent Med J Adv Res. 2015; 1: 1–7. Publisher Full Text\n\nRohleder N: Stimulation of systemic low-grade inflammation by psychosocial stress. Psychosom Med. 2014; 76(3): 181–9. PubMed Abstract | Publisher Full Text\n\nde Oliveira C, Watt R, Hamer M: Toothbrushing, inflammation, and risk of cardiovascular disease: results from Scottish Health Survey. BMJ. 2010; 340: c2451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHujoel PP, Lingström P: Nutrition, dental caries and periodontal disease: a narrative review. J Clin Periodontol. 2017; 44 Suppl 18: S79–S84. PubMed Abstract | Publisher Full Text\n\nMilward MR, Chapple I: The role of diet in periodontal disease. Clin Dent Heal. 2013; 52: 18–21. Reference Source\n\nEmerson SR, Sciarrillo CM, Kurti SP, et al.: High-Fat Meal–Induced Changes in Markers of Inflammation and Angiogenesis in Healthy Adults Who Differ by Age and Physical Activity Level. Curr Dev Nutr. 2018; 3(1): nzy098. Publisher Full Text\n\nCarmo CDS, Ribeiro MRC, Teixeira JXP, et al.: Added Sugar Consumption and Chronic Oral Disease Burden among Adolescents in Brazil. J Dent Res. 2018; 97(5): 508–514. PubMed Abstract | Publisher Full Text\n\nAdegboye AR, Christensen LB, Holm-Pedersen P, et al.: Intake of dairy products in relation to periodontitis in older Danish adults. Nutrients. 2012; 4(9): 1219–1229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan K, Hwang E, Park JB: Association between Consumption of Coffee and the Prevalence of Periodontitis: The 2008-2010 Korea National Health and Nutrition Examination Survey. PLoS One. 2016; 11(7): e0158845. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacedo RM, Brentegani LG, Lacerda SA: Effects of coffee intake and intraperitoneal caffeine on bone repair process--a histologic and histometric study. Braz Dent J. 2015; 26(2): 175–180. PubMed Abstract | Publisher Full Text\n\nYi J, Yan B, Li M, et al.: Caffeine may enhance orthodontic tooth movement through increasing osteoclastogenesis induced by periodontal ligament cells under compression. Arch Oral Biol. 2016; 64: 51–60. PubMed Abstract | Publisher Full Text\n\nMachida T, Tomofuji T, Ekuni D, et al.: Severe periodontitis is inversely associated with coffee consumption in the maintenance phase of periodontal treatment. Nutrients. 2014; 6(10): 4476–4490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZare Javid A, Seal CJ, Heasman P, et al.: Impact of a customised dietary intervention on antioxidant status, dietary intakes and periodontal indices in patients with adult periodontitis. J Hum Nutr Diet. 2014; 27(6): 523–532. PubMed Abstract | Publisher Full Text\n\nNielsen SJ, Trak-Fellermeier MA, Joshipura K, et al.: Dietary Fiber Intake Is Inversely Associated with Periodontal Disease among US Adults. J Nutr Nutr Epidemiol. 2016; 146(12): 2530–2536. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlavin J: Why whole grains are protective: biological mechanisms. Proc Nutr Soc. 2003; 62(1): 129–134. PubMed Abstract | Publisher Full Text\n\nWoelber JP, Bremer K, Vach K, et al.: An oral health optimized diet can reduce gingival and periodontal inflammation in humans - a randomized controlled pilot study. BMC Oral Health. 2016; 17(1): 28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHassan-Wassef H: Food habits of the Egyptians: newly emerging trends. East Mediterr Health J. 2004; 10(6): 898–915. PubMed Abstract\n\nWolosiak R, Worobiej E, Piecyk M, et al.: Activities of amine and phenolic antioxidants and their changes in broad beans (Vicia faba) after freezing and steam cooking. Int J Food Sci Technol. 2010; 45(1): 29–37. Publisher Full Text"
}
|
[
{
"id": "55206",
"date": "04 Nov 2019",
"name": "Reema Fayez Tayyem",
"expertise": [
"Reviewer Expertise Clinical and community nutrition."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study aimed to evaluate the prevalence and the severity of periodontal disease and its correlation with different risk factors.\nThe paper is well-written and presented paper. However, many concerns and questions should be addressed to be able to index this paper.\n\nAll the comments and suggestions are presented in the pdf attached.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5299",
"date": "12 Mar 2020",
"name": "Marwa Abbass",
"role": "Author Response",
"response": "Dear Reviewer:Thanks for your comprehensive revision for the current articleThese are the responses for your valuable commentsComment 1:“It should be among adults living in Cairo”Answer: When designing the study the following points were agreed by the authors:-Great Cairo encompasses more than 15 million, a large number of them are internal immigrants from different regions of Egypt.-low and middle SES Egyptians are the main beneficiaries of the free services at the Faculty of Dentistry, Cairo University which is a primary care center in Egypt. --To provide diversity, adults attending 3 private care centers also were recruited in the study to make sure of a heterogeneous population sample.The authors agree with the reviewer that the sample is a small convenient one and that the population at the great Cairo may differ from their companions in rural Upper Egypt areas. Therefore, the title has been modified by adding “a convenient sample of” to the adult Egyptian population as well as a limitation paragraph that has been added to the discussion section to represent the reviewer and the author’s point of view. Revised text:TitleThe occurrence of periodontal diseases and its correlation with different risk factors among a convenient sample of adult Egyptian population: a cross-sectional studyDiscussionMoreover, among the limitations of the present study is the small and convenient sample recruited from adults attending the free dental clinic at the Faculty of Dentistry and three private clinics at great Cairo. Although, a large number of great Cairo residences are internal immigrants from different regions of Egypt, including other geographical regions from Egypt may provide a better and accurate representation of the Egyptian population.Comment 2:“Is the present study population-based survey? Did the authors include patients from all the geographical areas in Egypt?”Answer:Refer to the answer of comment #1Revised text:Refer to the revised text of comment #1Comment 3:I don't think the authors really measured the prevalence of periodontitis. They rather estimated the occurrence of periodontitis among the conveniently selected group.Answer:The authors agree with the reviewer and appreciate this point of view, therefore, the statement “a convenient sample of” has been added to the title as well as the term “prevalence” has been substituted by the term “occurrence” in the whole manuscript. Revised Text:TitleThe occurrence of periodontal diseases and its correlation with different risk factors among a convenient sample of adult Egyptian population: a cross-sectional studyComment 4:“and crackers”Answer:Rewarding has been performed according to the reviewer's recommendations.Revised Text:Periodontitis was positively correlated with age, carbohydrates other than bread, grains, and crackers, as well as caffeinated drinksComment 5:“as well as”Answer:Rewarding has been performed according to the reviewer's recommendations.Revised Text:Periodontitis was positively correlated with age, carbohydrates other than bread, grains, and crackers, as well as caffeinated drinksComment 6:“can be defined or is defined”Answer:Rewarding has been performed according to the reviewer's recommendations. Revised Text:Periodontitis is defined as a chronic, progressive inflammatory disease affecting the periodontium surrounding the tooth.Comment 7:“what do you mean by this?”Answer:Following the reviewer’s recommendation, clarification for this point has been performed.Revised Text:Therefore, the aim of the present study is to investigate the incidence of periodontal diseases in correlation with the risk factors amongst a convenient sample from the Egyptian population.Comment 8:“If all the patients from Cairo, then how you generalize the results for all the Egyptian population?”Answer:When designing the study the following points were agreed by the authors:-Great Cairo encompasses more than 15 million, a large number of them are internal immigrants from different regions of Egypt.-low and middle SES Egyptians are the main beneficiaries of the free services at the Faculty of Dentistry, Cairo University which is a primary care center in Egypt.-To provide diversity, adults attending 3 private care centers also were recruited in the study to make sure of a heterogeneous population sample.The authors agree with the reviewer that the sample is a small convenient one. In the manuscript the sentence “Convenience sample was utilized in this study” has been used to describe the included sample.The authors modified the title by adding “a convenient sample of” to the adult Egyptian population as well as a limitation paragraph that has been added to the discussion section to represent the reviewer’s point of view. Revised Text:TitleThe occurrence of periodontal diseases and its correlation with different risk factors among a convenient sample of adult Egyptian population: a cross-sectional study DiscussionMoreover, among the limitations of the present study is the small and convenient sample recruited from adults attending the free dental clinic at Faculty of Dentistry and three private clinics at great Cairo. Although, a large number of great Cairo residences are internal immigrants from different regions of Egypt, including other geographical regions from Egypt may provide a better and an accurate representation of the Egyptian population.Comment 9:“I think this number of patients is not enough to say it is a population study!!!”Answer:Refer to answer of comment #8Revised Text:Refer to the revised text of comment #8Comment 10:“number”Answer:Rewarding has been performed according to the reviewer's recommendations.Comment 11:“were”Answer:Rewarding has been performed according to the reviewer's recommendations.Revised Text:In this study, positive correlations were found between calculus, periodontitis, and age."
},
{
"c_id": "5403",
"date": "27 Apr 2020",
"name": "Reema Tayyem",
"role": "Reviewer Response",
"response": "All the requested corrections are addressed in a satisfactory manner."
}
]
},
{
"id": "56359",
"date": "26 Nov 2019",
"name": "Enas Elgendy",
"expertise": [
"Reviewer Expertise Periodontology & Oral medicine"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study in the field of periodontology as the authors have tried to find the correlation between the common risk factors of periodontitis and the different stages of the diseases among Egyptian patients according to the new classification published in 2018.\n\nHowever, the authors have excluded patients with aggressive periodontitis without justification for their exclusion and this group of patients represents most of patients in stages 3 & 4.\nThe results of the study were low in stage III & VI (4.4% & 2.05 % respectively) and this may be attributed to excluding aggressive periodontitis from the study design. The aggressive periodontitis should be included in the study design because this type with sever chronic periodontitis represent stage III &VI.\n\nIn addition, the study design did not include diabetes mellitus and smoking among the studied risk factors. One of the strength points of the study is that the national common food and drinks as beans and tea were included among the risk factors.\nThis study has not been conducted before in Egyptian patients according to stages and the finding of different risk factors among the Egyptian patients can reduce the prevalence of these diseases in the future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "5300",
"date": "12 Mar 2020",
"name": "Marwa Abbass",
"role": "Author Response",
"response": "Dear reviewer:Thanks for your comprehensive revision of this article, we do really appreciate your comments.These are our responses for your valuable commentsComment 1:“However, the authors have excluded patients with aggressive periodontitis without justification for their exclusion and this group of patients represents most of the patients in stages 3 & 4.The results of the study were low in stage III & VI (4.4% & 2.05 % respectively) and this may be attributed to excluding aggressive periodontitis from the study design. The aggressive periodontitis should be included in the study design because this type with severe chronic periodontitis represents stage III &VI. Answer:The authors do appreciate the reviewer’s comment. This is the first study to be conducted using the new periodontal classification, therefore this point despite its importance has not been taken into consideration as usually in previous studies following the old classification the aggressive periodontitis patient was excluded. A limitation paragraph has been added in the discussion section to clarify this valuable point for further research implementation.Revised Text:An important limitation of the current work is the exclusion of patients with aggressive periodontitis where this group of patients together with the severe chronic periodontitis patients represents the individuals in stages III &VI. The low recorded percentages of periodontitis in these two stages could be referred to as this exclusion. This consideration should be taken in future studies implementing the new 2018 periodontal classification.Comment 2:“In addition, the study design did not include diabetes mellitus and smoking among the studied risk factors. One of the strongest points of the study is that the national common food and drinks as beans and tea were included among the risk factors.”Answer:The authors agree with the reviewer’s comment and because of this in the discussion section the following statement was written to clarify this “Even though the current work investigated the occurrence of periodontal diseases in correlation to different risk factors, important risk factors still need to be investigated such as smoking and glycosylated haemoglobin level.” Sincerely,Marwa Abbass"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1740
|
https://f1000research.com/articles/8-1597/v1
|
05 Sep 19
|
{
"type": "Research Article",
"title": "Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines",
"authors": [
"Adriana E. Kajon",
"Xiaoxin Li",
"Gabriel Gonzalez",
"Susan Core",
"Helga Hofmann-Sieber",
"Shuguang Leng",
"Xiaoxin Li",
"Gabriel Gonzalez",
"Susan Core",
"Helga Hofmann-Sieber",
"Shuguang Leng"
],
"abstract": "Background: The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig (Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.",
"keywords": [
"Mastadenovirus",
"guinea pig",
"cell culture",
"PCR"
],
"content": "Introduction\n\nAdenovirus (AdV)-associated pneumonia has been widely recognized as a cause of morbidity and occasional death in laboratory guinea pig (GP, Cavia porcellus) colonies worldwide since the early 1980s (Brennecke et al., 1983; Crippa et al., 1997; Eckhoff et al.,1998; Feldman et al., 1990; Finnie et al., 1999; Naumann et al., 1981; Shankar, 2008). However, knowledge about the alleged causative agent, guinea pig adenovirus (GPAdV), and the pathogenesis of the documented disease is still very limited. Published cases document infections of low morbidity and variable susceptibility, likely as a result of host age differences, immune competence and/or strain differences. The lack of adequate in vitro systems to isolate and propagate GPAdV has precluded its formal characterization to allow for the development of diagnostic assays and reagents, and for the execution of complex pathogenesis and basic virology studies. The only molecular data currently available for GPAdV in public databases is limited to a 234-nucleotide sequence of the hexon gene contributed by Pring-Akerblom and colleagues in 1997.\n\nIn this paper we report the development of a novel GP tracheal epithelial cell line, the feasibility of virus propagation of GPAdV in this and other commercially available GP cell lines, and the preliminary genetic characterization of two virus strains isolated from infected animal tissue originating from two different continents.\n\n\nMethods\n\nGPAdV-infected lung tissue originating from a 1996 cluster of pneumonia cases in a laboratory GP (strain not specified) colony in Melbourne, Australia (Finnie et al., 1999) was obtained as a generous gift from the Veterinary Services Division, Institute of Medical and Veterinary Science to author AEK. Nasal scrapings from outbred Ibm:GOHI GPs (specific pathogen-free, Biological Research Laboratories Ltd., Füllinsdorf, Switzerland) experimentally infected with lung homogenates of GPs diagnosed with bronchopneumonia during a colony outbreak in Prague, Czech Republic in 1996 (Butz et al., 1999) were obtained as a gift from the Wolf laboratory at the Section of Comparative Medicine, Yale University School of Medicine to author AEK.\n\nGP (Cavia porcellus) cell lines JH4 clone 1 (lung fibroblasts; RRID: CVCL_4299, catalog # CCL-158), 104-C1 (fetal fibroblasts; RRID: CVCL_2267, catalog # CRL-1405), and GPC-16 (colorectal adenocarcinoma; RRID: CVCL_3463, catalog # CCL-242) were purchased from ATCC and maintained and propagated as recommended by the vendor: JH4 clone 1 cells were grown in Hams’s F12K medium supplemented with 10% Fetal Bovine Serum (FBS); 104 C1 cells were grown in RPMI 1640 supplemented with 10% FBS; and GPC-16 cells were grown in Minimum Essential Medium (MEM) Eagle supplemented with 10% FBS. For infection, the FBS content was reduced to 2%. Cell monolayers were photographed in an Olympus CKX41 inverted microscope (Olympus Corporation) equipped with an Infinity 2 camera (Teledyne Lumenera).\n\nNo live animals were involved in the study. We obtained the tracheas from two 2-month old male Hartley GPs that had been euthanized by intraperitoneal injection of a lethal dose of sodium pentobarbital after being used for laboratory animal technician training under Lovelace Respiratory Research Institute (LRRI) Institutional Animal Care and Use Committee (IACUC)-approved protocols. The tracheas were dissected at LRRI’s necropsy core and collected in 1X phosphate-buffered saline (PBS) for transportation to the Kajon laboratory.\n\nThe two tracheas were sliced longitudinally, the epithelial surface was scraped using a convex surgical scalpel, and the scraped material from both tracheas was pooled and collected into a 50mL conical tube containing sterile wash medium [1X PBS and 2% penicillin/streptomycin (GIBCO/ThermoFisher Scientific)]. The cell suspension was centrifuged at 800rpm for five minutes at 4°C with no break. The supernatant was aspirated off and the pellet was resuspened in 10mL of wash medium. This process was repeated twice. After the final wash, the cell pellet was resuspened in 12mL of growth medium [50% DMEM and 50% Ham's F-12 supplemented with 15mM HEPES (Invitrogen/ThermoFisher Scientific), 3.6mM sodium bicarbonate (Sigma), 2mM L-Glutamine (Invitrogen/ ThermoFisher Scientific), 5µg/ml insulin (Lonza), 5µg/ml transferrin (Lonza), 20ng/ml cholera toxin (Lonza), 12.5ng/ml epidermal growth factor (EGF, Lonza), 30ug/mL Bovine Pituitary Extract (BPE, Lonza), 0.01µM retinoic acid (Lonza), and 5% DMSO (Fluka 41639)]. In order to select against the fibroblast population present in the primary cell preparations, the culture medium contained no serum. A fraction of the cells were plated for propagation for two to three passages and the rest of the primary cell preparation was alliquoted into cryovials, frozen at -80°C and then moved for long term storage to liquid nitrogen.\n\nFor propagation in preparation for transfection, frozen cells at passage three were thawed and centrifuged at 800rpm for five minutes at 4°C with no break with 10mL growth medium to remove DMSO. Cells were then resuspended in 10mL of growth medium for plating onto a collagen-coated 96-well plate (Biocoat™ Matrigel™, catalog #354607, BD) and maintained at 37°C and 5% CO2. Twenty days after plating, monolayers were transfected with mammalian expression plasmid pRSVneoT, a generous gift of Dr. Jim Pippas (University of Pittsburgh) using PolyMag Transfection Reagent (OZ Biosciences USA, San Diego, CA), and Magnetofection™ protocols recommended by the manufacturer. Briefly, pRSVneoT DNA was diluted in serum free medium and mixed with PolyMag™ transfection reagent (catalog # PN30100) in various proportions in order to deliver 200ng, 500 ng or 1ug of plasmid DNA per well in 180 ul. Mixes were incubated for 20 minutes at room temperature; the transfection complexes were then added directly to cells, and the culture plate was placed on a 96-magnets plate (catalog # MF10096). for 20 minutes. The magnetic plate was then removed, and the cells were cultured overnight. The next day, the culture medium was replaced with fresh medium containing 0.4mg/ml of G418. The transfected cells were kept in culture for four weeks and monitored for proliferation.\n\nA single viable clone was recovered and further propagated to establish a cell line. The line was frozen at several stages of serial passaging (passages three, five and six) in liquid nitrogen for long term preservation. After five initial passages in nutrient rich medium, the line was adapted to grow in Eagle Minimum Essential Medium (MEM) supplemented with 8% New Born Calf Serum (NBCS), L-glutamine, HEPES 25mM, 7.5% Sodium Bicarbonate, and penicillin/streptomycin, without G418. For infection, the NBCS content was reduced to 2%.\n\nChromosomes were prepared, fixed, and stained following a modification of the protocol used by Costa et al. (2005). Briefly, cells at passage 10 were grown in a 75cm2 flask. When the cell confluence reached 80-90%, Colcemid™ (N-methyl-N-deacetyl-colchicine, Sigma-Aldrich) was added to the cultures at a final concentration of 0.06mg/ml to arrest cells in metaphase. Two hours later, cells were harvested by trypsin digestion and then subsequently treated with 0.075M KCl hypotonic solution for 30 minutes at 37°C and then fixed with methanol-acetic acid 3:1 at 4°C overnight. Air dried slides were stained with Giemsa. A total of 105 well-spread metaphases were examined by light microscopy under an oil immersion objective to count the number of chromosomes per metaphase.\n\nThe GPTEC-T line at passage 25 was submitted to Leibniz Institute DSMZ, Braunschweig, Germany for authentication services. Cytochrome c oxidase subunit 1 (CO1) DNA bar coding (Hebert et al., 2003) was performed following institutional standard operating procedures based on the protocols described by Ivanova and colleagues (Ivanova et al., 2012). Detection of the SV40 T antigen coding sequence by PCR was carried out on total DNA extracted from cell suspensions using QIAamp DNA Micro Kit (catalog # 56304, QIAGEN, Valencia, CA) using primers F-SV-40 5’-GACTACAAGGATGACGATGACAAGCTC-3’ and R-SV-40 5’-CTCCACACAGGCATAGAGTGTCTG-3’. Cell lines from the DSMZ collection, HeLa (DSMZ ACC 057) and 293 (DSMZ ACC 305), and 104C1 (ATCC CRL-1405) were used as negative controls. Cell line 293T (DSMZ ACC 635) was used as a positive control for the presence of the SV40 T antigen coding sequence.\n\nCell monolayers on cover slips were fixed using 4% paraformaldehyde, followed by quenching using 10mM glycine and permeabilization with 0.5% Triton X-100. Cells were washed and blocked for one hour in 2% normal goat serum. Staining for epithelial cell marker ZO-1 was carried out with ZO-1 rabbit polyclonal antibody (RRID: AB_2533938, catalog # 61-7300, Zymed/ ThermoFisher Scientific) and Cyanine Cy™3-conjugated goat anti-rabbit polyclonal secondary antibody (RRID: AB_2338000, catalog # 111-165-003, Jackson ImmunoResearch;). Co-staining for SV40 T antigen was carried out with mouse monoclonal PAb416 (Abcam; RRID: AB_302561) and Cyanine Cy™5-conjugated goat anti-mouse polyclonal secondary antibody (RRID: AB_2338715, catalog # 115-175-205, Jackson ImmunoResearch). Nuclei were counterstained with Hoechst 33342 (Pierce/ ThermoFisher Scientific). Images were obtained at a 40x magnification in a Zeiss Axioskop epifluorescence microscope using Slide Book 6 digital microscopy software (Intelligent Imaging Innovations, Inc.).\n\nA modification of the PCR protocol developed by Pring-Åkerblom & colleagues (1997) was used to test the original materials obtained as sources of GPAdV, and to monitor virus propagation and serial passaging in cell culture. Lung homogenate supernatants, the nasal scraping suspension and infected cell lysates were processed for total DNA extraction using QIAamp DNA Micro Kit (catalog #56304, QIAGEN, Valencia, CA). A total of 10μl of QIAgen column-eluted DNA was added to each 40μL reaction mixture containing 50mM KCl, 10mM Tris-HCl (pH 9.0), 1.5mM MgCl2, 0.1% Triton X-100, 0.2mM of each dNTP, 0.2μM of each GPAdV-specific primer (GPAdV1: 5'-GCCAGGAGGCGGTAGAC-3' and GPAdV2: 5'-CCAAGACGCGATTGTCT-3'), and 1.25U of GoTaq® DNA polymerase (catalog # M3001, Promega Corporation, Madison, WI) following manufacturer-recommended thermal cycling conditions for GoTaq® DNA polymerase-mediated PCR (an initial step of denaturation at 95°C for two minutes, 35 to 50 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds; the final cycle had a prolonged extension time of five minutes). PCR products were analyzed by horizontal gel electrophoresis in 1% GeneMate LE agarose (catalog # E-3120-500, BioExpress/VWR, Radnor, PA) gels, prepared and run in 1X TBE buffer (0.09 M Tris-borate, 2 mM EDTA, pH 8.0). Gels were stained with ethidium bromide and visualized and photographed in a Gel-Doc imaging system (Bio-Rad, Hercules, CA). To control for the specificity of the reaction in the absence of positive controls, amplicons were submitted for Sanger sequencing with the amplification primers to Genewiz (South Plainfield, NJ).\n\nThe sequences of the complete hexon gene, partial DNA polymerase gene and partial penton base gene were determined for the preliminary characterization of virus isolates, and for confirmation of their identity as GPAdV. Degenerate primers spanning conserved regions based on analysis of available mammalian adenovirus sequences were designed to generate the initial amplicons. Degenerate primers were designed based on the alignment and analysis of a variety of known mastadenovirus hexon genes, upstream pVI genes, downstream protease genes, DNA pol genes and penton base genes. EcoRI or BamHI extension sites were incorporated to the 5’ end of these degenerate primers to facilitate cloning for subsequent sequencing. PCR products were gel-purified, digested with EcoRI and BamHI (Promega), and cloned into EcoRI-BamHI-digested and Calf Intestinal Alkaline Phosphatase (Promega Corporation, Madison, WI) -treated pUC19. Plasmid DNA was purified with GenEluteTM Plasmid Miniprep Kit (catalog # PLN350-1KT Sigma) and sequenced with primers pUC19FW (5‘-TGCAAGGCGATTAAGTTGGG-3‘) and pUC19RV (5‘-CTTCCGGCTCGTATGTTGTG-3‘). PCR products obtained with other primers were sequenced directly using the specific amplification (or other appropriate) primers. Sanger sequencing services were contracted from Genewiz (South Plainfield, NJ). All primers used in this study were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). A complete list of all primers used for the amplification and sequencing of the three regions of the GPAdV genome is provided in Table 1. PCR was carried out in a total volume of 50 µl with nuclease free water, PCR primers at a final concentration of 0.5 µM, 100–200 ng of total DNA, dNTP at a final concentration of 0.2 mM, 1 x iProof HF Buffer, 0.5 µl of High-Fidelity iProof DNA Polymerase (catalog # 1725300, Bio-Rad, Hercules, CA). Alternatively, for longer amplicons or difficult template regions PCR was also carried out using TaqSelect 2X Premix (catalog # 30041, Lucigen Corporation) containing dNTP, buffer and high-fidelity TaqSelect DNA Polymerase. PCR cycling conditions for product amplification varied with the primer pair and DNA polymerase (TaqSelect or iProof) but followed the corresponding manufacturers’ recommendations. The initial denaturation was carried out at 94°C for two minutes when using TaqSelect and at 98°C for 30 seconds when using iProof. When using TaqSelect, annealing temperatures were set at 2°C below the lowest calculated melting temperature (Tm). When using iProof, primers longer than 30 nt were annealed at 3°C above the lowest Tm for 30 seconds and primers 20 nt or shorter were annealed at a temperature equal to the lowest Tm for 30 seconds. The extension step was carried out at 72 °C for one minute/kb for TaqSelect, and 15 seconds/Kb for iProof. The initial denaturation step was followed by 35 cycles of [denaturation-annealing-extension] and an extra five minutes at 72°C for final extension and holding at 4°C.\n\n¥ Feldman et al., 2001; ^ Pring-Akerblom et al., 1997\n\nSequence reads were edited in EditSeq 15 (Lasergene® Molecular Biology Suite, DNASTAR) to remove vector sequences in and poor quality sections. Contigs were assembled in Seqman Pro 15 (Lasergene® Molecular Biology Suite, DNASTAR). Sequences for the complete hexon gene and partial DNA polymerase and penton base genes were deposited in GenBank under accession numbers MK787233-MK787238 (see Underlying data).\n\nMultiple sequence alignments (MSAs) were prepared with partial or complete sequences of GPAdV and homologous sequences in other mastadenovirus species of corresponding to loci encoding the DNA polymerase, penton base and hexon. The species D Fowl aviadenovirus 764 (KT862811 FadV-D 764) was used to root the adenovirus species isolated from mammals. Adenoviral species isolated from mammals considered for these comparisons were: AB026117 Porcine adenovirus 3 (PAdV-3), NC_034834 Deer adenovirus B (Odocoileus hemionus adenovirus, OdAdV-B), AF252854 Bovine adenovirus 2 (BAdV-2), AF030154 Bovine adenovirus 3 (BAdV-3), AC_000003 Canine adenovirus 1 (CAdV-1), X73487 Human adenovirus A12 (HAdVA-12), AY594255 Human adenovirus B7 (HAdV-B7), AC_000008 Human adenovirus C5 (HAdV-C5), KT862547 Human adenovirus D8 (HAdV-D8), AP014853 Human adenovirus E4 (HAdV-E4), KU162869 Human adenovirus F40 (HAdV-F40), DQ923122 Human adenovirus G52 (HAdV-G52), NC_000942 Murine adenovirus A (MAdV-A), EU835513 Murine adenovirus 3 (MAdV-3), KP329564 Simian adenovirus 16 (SAdV-16), FJ025929 Simian adenovirus 47 (SAdV-47), HQ241820 Simian adenovirus 50 (SAdV-50), KX505867 Simian adenovirus (SAdV WIV19), HQ913600 Titi monkey adenovirus (TMAdV), KT013209 Cynomolgus Adenovirus (CynAdV), MF198459 Rhesus adenovirus 65 (SAdV-65), AF258784 Tree shrew adenovirus 1 (TsAdV-1), JN252129 Bat adenovirus 2 (BtAdV-2), KT698853 Bat adenovirus WIV9 (BtAdV-WIV9), and JN418926 Equine adenovirus 1 (EadV-1).\n\nMSAs were built with MAFFT v7.429 (Katoh & Standley, 2016) using the FFT-NS-i algorithm. The phylogenetic relation of both GPAdV were explored with Bayesian and maximum likelihood methods with MrBayes v3.2.6 (Ronquist et al., 2012) and RAxML v8.2.10 (Stamatakis, 2014), respectively. In both cases, the general time-reversible substitution model with a gamma-modeled heterogeneity among sites and allowing for invariant sites (GTR+I+G), was used for the phylogenetic inference. The substitution model was chosen using the best Aikaike information criterion corrected (AICc) inferred with jmodeltest 2 v0.1.10 (Darriba et al., 2012). MrBayes was executed with five million states per chain to assure convergence. The support in branches for the topology inferred by RAxML was calculated with bootstrap on 5000 repetitions.\n\n\nResults\n\nA novel GP epithelial cell line was generated by transfection of primary tracheal epithelial cells from a Hartley male GP with plasmid pRSVneoT as described in the Methods section. The new cell line designated GPTEC-T to denote its origin and features.\n\nGPTEC-T cells at passage 23 were karyotyped at LRRI. While the diploid number of chromosomes for Cavia porcellus is 64, data from the examination of 105 GPTEC-T metaphases demonstrated aneuploidy, showing a distribution of 56 - 81 chromosomes with 60 and 61 chromosomes found in 20 and 19% of the metaphases (Figure 1A; Kajon et al., 2019).\n\nA. Chromosome numbers in metaphases of GPTEC-T cells at passage 23. A total of 105 metaphases were examined by light microscopy. B. Detection of T antigen coding sequence by PCR for quality control at DSMZ, Germany. Total DNA was extracted from GPTEC-T and various other cell lines included in the test as controls, and used as a template. M: Molecular marker Generuler 1kB plus (Thermo Fisher Scientific). C. Indirect immunofluorescence staining of GPTEC-T cells for ZO-1 and SV40 T antigen. Cells were stained with primary antibodies anti ZO-1 and anti SV40 T antigen and appropriate secondary antibodies as described in the Methods section. Nuclei were stained with Hoechst. Images were acquired with a Zeiss Axioskop epifluorescence microscope at 40X magnification. Merged image of ZO-1 staining at the plasma membrane (green) and nuclear staining for T antigen (red) and Hoechst (blue).\n\nThe GPTEC-T line was further authenticated by CO1 DNA bar coding as of Cavia porcellus origin at Leibniz Institute DSMZ and confirmed to encode SV40 T antigen coding region by PCR (Figure 1B). As shown in Figure 1C, examination of GPTEC-T cells by indirect immunofluorescence demonstrated that the line expresses epithelial marker ZO-I and, as expected, SV40 T antigen.\n\nGPTECT-T, GPC-16, JH4 and 104-C1 cell monolayers in 24-well plates were infected with 50 μl of lung homogenate supernatant, or 50 μl of nasal scraping and monitored for the development of cytopathic effect (CPE) for 7–14 days. PCR screening of original specimens and their passages in GP cell lines yielded the expected 281bp fragment for all tested samples (Figure 2). The identity of the amplified products was verified by Sanger sequencing. Representative amplicon sequences were deposited in GenBank under accession numbers MN250852 and MN250853. The original nasal scraping sample did not test positive by PCR, but passage one in culture did, demonstrating the presence of infectious virus in the original material. Amplicon sequences were 98–100% identical to the sequence submitted to GenBank by Pring-Åkerblom and colleagues under accession number X95630 (Guinea pig adenovirus hexon). CPE of variable magnitude was observable in monolayers infected with both sources of virus but not in the controls. The isolated strains were designated AUS96 and CZE96. In all tested cell lines, CPE was initially focal but increased in magnitude in passages two and three. Representative images from cell monolayers infected with PCR-positive passage three of AUS96 or CZE96 in the corresponding lines are shown in Figure 3.\n\nTotal DNA was extracted from GP lung homogenate, nasal scrapings or cell culture aliquots with Qiagen DNA micro kit (QIAGEN, Valencia, CA) and used as a template for PCR amplification of a portion of the hexon gene using the primers and protocol developed by Pring-Akerblom et al. (1997). Ch-Ext. LH: chloroform-extracted lung homogenate supernatant; AUS 1 and AUS 2: designations for two different Australian lung tissue samples; CZE NS: nasal scraping suspension from GPs experimentally infected with Czech Republic strain; C: negative control; M. EZ Load™ 100 bp Molecular Ruler (Bio-Rad, Hercules, CA); p1: first passage in culture from original sample.\n\nInfected cultures and uninfected controls were examined daily for the occurrence of cytopathic effect. Photographs were taken at various days post infection (dpi) as indicated at 20X magnification in an Olympus CKX41 inverted microscope (Olympus Corporation) equipped with an Infinity 2 camera (Teledyne Lumenera).\n\nThe complete hexon gene sequence and partial sequences for the penton base gene and DNA polymerase gene were successfully determined for both virus strains using our strategy of an initial round of amplification using degenerate primers followed by primer walking to obtain readings from both strands. We obtained partial sequence data for 789 bases and 801 bases of the DNA polymerase gene, and partial sequence data for 473 bases and 549 bases of the penton base gene for strains AUS96 and CZE96, respectively. Sequences were deposited in GenBank under accession numbers MK787233-MK787238.\n\nWhile the partial sequences for the DNA polymerase gene were identical between both isolated strains, a few nucleotide differences were found between strains AUS96 and CZE96 in the hexon gene and partial penton base gene sequences (available as Underlying data; Kajon et al., 2019).\n\nOur preliminary phylogeny reconstructions for GPAdV based on partial nucleotide sequences for the DNA polymerase, and penton base genes and on complete hexon gene sequences are presented in Figure 4 A–C. The three trees show that the virus is a novel mastadenovirus genetically distinct from other species in the genus. A relatively high GC content of 59.30% and 59.34% was found in the hexon gene sequences of strains AUS96 and CZE96, respectively (Kajon et al., 2019). A comparison of GC contents among the examined mastadenoviruses is shown in Figure 5.\n\nSequences obtained for the DNA polymerase (partial), penton base (partial) and hexon genes for the two isolated GPAdV strains were aligned with sequences for the corresponding regions of the genome of twety five selected mastadenoviruses in MAFFT using the FFT-NS-i algorithm. The phylogenetic relationships of both GPAdV strains were explored with Bayesian and maximum likelihood methods with MrBayes and RAxML, respectively. The maximum likelihood-inferred phylogenetic trees were rooted with the sequence of species D Fowl aviadenovirus 764 (KT862811 FAdVD 764). Branches are annotated with the bootstrap support (RAxML) and the posterior probability (MrBayes). The absense of posterior probability in the Bayesian topology is marked with a dash (-). Tip names from adenoviral species isolated in human, murine, and guinea pig hosts, are colored in blue, green and red, respectively. The scale represents 0.25 nucleotide mutations per site. A. Phylogenies inferred using the partial nucleotide sequence of the DNA polymerase gene. B. Phylogenies inferred using the partial nucleotide sequence of the penton base gene. C. Phylogenies inferred using the nucleotide sequence of the complete hexon gene.\n\nThe percentage of cytosine and guanine (%GC) per sequence was calculated for the hexon gene sequences (blue) and for available complete genomic sequences (blue) as context. The vertical axis represents the % GC and the compared mastadenovirus sequences are shown in the horizontal axis on descending order according to the %GC in the hexon gene.\n\n\nDiscussion\n\nThe lack of suitable culture systems for the isolation and propagation of GPAdV has been a major impediment in the development of this virus as a potential model system for the study of adenoviral pathogenesis. This study established the feasibility of propagating, albeit at an apparent low efficiency, GPAdV in cultures of immortalized lines of GP cells of a variety of types. This important matter warrants further investigation and efforts to establish a robust culture platform for virus stock production and titration. In addition, the availability of a variety GP cell lines and molecular assays to support and monitor viral replication will be critical for the study of virus-host interactions in vitro.\n\nOur initial culture efforts recovered enough viral DNA to generate sequence data for the complete hexon gene and partial sequence data for the DNA polymerase and penton base coding regions for both isolated strains, AUS96 and CZE96.\n\nOur sequence data and analysis complement the findings of Pring-Åkerblom and colleagues’ (1997) and Feldman and colleagues’ (2001) in providing strong evidence that GPAdV is a unique species in the genus Mastadenovirus. Interestingly, our data also show the two strains isolated from infected GPs in geographically distant locations to be surprisingly closely related.\n\nThe topology of the tree generated in this study for the complete hexon gene is similar to the one reported by Feldman and colleagues (2001) using a 534 amino acid sequence inferred from a 5’ partial 1603 nucleotide sequence of the hexon gene. The original source of total DNA used as a template for PCR amplification by these investigators was lung tissue obtained from two cases of fatal pneumonia documented in the United States in 1997. Unfortunately, this sequence was never submitted to Genbank and could not be incorporated into our alignment for comparison.\n\nThe generation and analysis of whole genome sequences (WGS) will provide additional data for a comprehensive description of the genetic organization of the GPAdV genome and for a better assessment of genetic diversity between the two isolated strains. The high GC content identified for the hexon gene relative to that reported for other mammalian adenoviruses is intriguing and highlights the need for further in-depth analysis once the WGSs are available for these and other GPAdV strains. The GC content has been shown to vary widely among AdV genomes and to correlate with genome size and CpG representation (Davison et al., 2003; Tan et al., 2017).\n\n\nData availability\n\nGPAdV strain AUS96 complete hexon gene sequence on Genbank, Accession number MK787233\n\nGPAdV strain CZE96 complete hexon gene sequence on Genbank, Accession number MK787234\n\nGPAdV strain AUS96 partial DNA polymerase gene sequence on Genbank, Accession number MK787235\n\nGPAdV strain CZE96 partial DNA polymerase gene sequence on Genbank, Accession number MK787236\n\nGPAdV strain AUS96 partial penton base gene sequence on Genbank, Accession number MK787237\n\nGPAdV strain CZE96 partial penton base gene sequence on Genbank, Accession number MK787238\n\nGPAdV strain AUS96 partial hexon gene amplicon sequence on Genbank, Accession number MN250852\n\nGPAdV strain CZE96 partial hexon gene amplicon sequence on Genbank, Accession number MN250853\n\nZenodo: Isolation and Initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines. http://doi.org/10.5281/zenodo.3356997 (Kajon et al., 2019)\n\nThis project contains the following underlying data:\n\n- 104C1p48 CZE 14dpi.jpg (raw, unedited image showing CPE in a CZE96-infected 104-C1 cell culture from Figure 3)\n\n- 104C1p48 uninfected control 14dpi.jpg (raw, unedited image showing an uninfected 104-C1 cell culture control monolayer from Figure 3)\n\n- GPC-16 AUS96 5dpi.jpg (raw, unedited image showing CPE in an AUS96-infected GPC-16 cell culture from Figure 3)\n\n- GPC-16 p57 uninfected control 5dpi.jpg (raw, unedited image showing an uninfected GPC-16 cell culture control monolayer from Figure 3)\n\n- GPTEC-T AUS96 6 dpi.jpg (raw, unedited image showing CPE in an AUS96-infected GPTEC-T cell culture from Figure 3)\n\n- GPTEC-T p9(12) uninfected control 6 dpi.jpg (raw, unedited image showing an uninfected GPTEC-T cell culture control monolayer from Figure 3)\n\n- JH4 p42 Aus 96 11 dpi.jpg (raw, unedited image showing CPE in an AUS96-infected JH4 cell culture from Figure 3)\n\n- JH4 p42 control 11 dpi.jpg (raw, unedited image showing an uninfected JH4 cell culture control from Figure 3)\n\n- GPTEC-T IF 40X Air Dapi.tif (raw, unedited immunofluorescence image showing Hoescht staining only)\n\n- GPTEC-T IF 40X Air MERGED ZO-1 T antigen Dapi.tif (raw, unedited immunofluorescence image showing merged DAPI - ZO-1 and T antigen staining)\n\n- GPTEC-T IF 40X Air T antigen.tif (raw, unedited immunofluorescence image showing T antigen staining only)\n\n- Shuguang metaphase analysis.xls (raw karyotype counts underlying karyotype analysis)\n\n- smcgrath 2008-02-06 PCR screening.jpg (raw, unedited gel image from Figure 2)\n\n- Wilkens(E1)_170820181 GPTEC-T cell line authentication.pdf (results of DNA bar coding carried out by Leibniz Institute DSMZ)\n\n- DSMZ SV40 T antigen PCR assay (GPTEC-T cell line testing for SV40 T antigen at DSMZ, original unedited gel obtained from DSMZ with the report)\n\n- GC content values from Gabriel Gonzalez.xls (raw data used in the generation of Figure 5)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThe authors wish to thank the Veterinary Services Division, Institute of Medical and Veterinary Science, South Australia and the Wolf laboratory at the Section of Comparative Medicine, Yale University School of Medicine for their generous gift of the infected GP tissues used as a source of infectious virus in this study.\n\n\nReferences\n\nBrennecke LH, Dreier TM, Stokes WS: Naturally occurring virus-associated respiratory disease in two guinea pigs. Vet Pathol. 1983; 20(4): 488–91. PubMed Abstract | Publisher Full Text\n\nButz N, Ossent P, Homberger FR: Pathogenesis of guinea pig adenovirus infection. Lab Anim Sci. 1999; 49(6): 600–4. PubMed Abstract\n\nCosta UM, Reischak D, da Silva J, et al.: Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen. J Virol Methods. 2005; 128(1–2): 72–8. PubMed Abstract | Publisher Full Text\n\nCrippa L, Giusti AM, Sironi G, et al.: Asymptomatic adenoviral respiratory tract infection in guinea pigs. Lab Anim Sci. 1997; 47(2): 197–9. PubMed Abstract\n\nDarriba D, Taboada GL, Doallo R, et al.: jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8): 772. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavison AJ, Benko M, Harrach B: Genetic content and evolution of adenoviruses. J Gen Virol. 2003; 84(Pt 11): 2895–908. PubMed Abstract | Publisher Full Text\n\nEckhoff G, Mann P, Gaillard E, et al.: Naturally Developing Virus-Induced Lethal Pneumonia in Two Guinea Pigs (Cavia porcellus). Contemp Top Lab Anim Sci. 1998; 37(1): 54–7. PubMed Abstract\n\nFeldman SH, Richardson JA, Clubb FJ Jr: Necrotizing viral bronchopneumonia in guinea pigs. Lab Anim Sci. 1990; 40(1): 82–3. PubMed Abstract\n\nFeldman SH, Sikes RA, Eckhoff GA: Comparison of the deduced amino acid sequence of guinea pig adenovirus hexon protein with that of other mastadenoviruses. Comp Med. 2001; 51(2): 120–6. PubMed Abstract\n\nFinnie JW, Noonan DE, Swift JG: Adenovirus pneumonia in guinea pigs. Aust Vet J. 1999; 77(3): 191–2. PubMed Abstract | Publisher Full Text\n\nHebert PD, Cywinska A, Ball SL, et al.: Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270(1512): 313–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIvanova NV, Clare EL, Borisenko AV: DNA barcoding in mammals. Methods Mol Biol. 2012; 858: 153–82. PubMed Abstract | Publisher Full Text\n\nKajon AE, Li X, Gonzalez G, et al.: Isolation and Initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines. F1000Res. 2019. Publisher Full Text\n\nKatoh K, Standley DM: A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics. 2016; 32(13): 1933–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaumann S, Kunstýr I, Langer I, et al.: Lethal pneumonia in guinea pigs associated with a virus. Lab Anim. 1981; 15(3): 235–42. PubMed Abstract | Publisher Full Text\n\nPring-Åkerblom P, Blazek K, Schramlová J, et al.: Polymerase chain reaction for detection of guinea pig adenovirus. J Vet Diagn Invest. 1997; 9(3): 232–6. PubMed Abstract | Publisher Full Text\n\nRonquist F, Teslenko M, van der Mark P, et al.: MrBayes 3.2: efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst Biol. 2012; 61(3): 539–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShankar BP: Adenovirus Infection in Guinea Pig - A Case Study. Veterinary World. 2008; 1(9): 280. Reference Source\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan B, Yang XL, Ge XY, et al.: Novel bat adenoviruses with low G+C content shed new light on the evolution of adenoviruses. J Gen Virol. 2017; 98(4): 739–48. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "53516",
"date": "18 Sep 2019",
"name": "Márton Z. Vidovszky",
"expertise": [
"Reviewer Expertise adenovirology",
"phylogeny",
"molecular biology",
"animal virology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the isolation of two strains of guinea pig adenoviruses and their successful propagation on a newly developed and on three commercially available guinea pig (GP) cell lines. The manuscript is very relevant, as every successful, novel virus isolation is very important for adenovirology. The manuscript also discusses the partial phylogenetic comparison of adenoviral sequences with the examined AdV strains (full hexon gene and partial polymerase and penton base sequence parts).\n\nThe Introduction is thorough enough and dense in a good meaning. It only deals with guinea pig AdVs, but all the known publications are well collected.\n\nThe Methods section is a bit too detailed and long compared to the other parts of the manuscript. Sometimes the process is already published somewhere, and the manuscript refers to it correctly but also details shortly. This is unnecessary in some cases in my opinion.\n\nThe process of the primary cell line production and its immortalization is very well written. It is easy to follow the process and very interesting.\n\nThe successful isolation and propagation of the two GPAdVs are a very nice job and well written. The pictures of CPEs on different cell lines represent the changes correctly and well visualize this part of the research. The surprising parts of the adenoviral PCR detection in the homogenates and infected cell cultures are that the original nasal scraping sample was PCR negative for AdV, while the cell culture infected by this material showed CPE and it was PCR positive. I would be curious of the opinion of the authors, how this can be possible, i.e., that the PCR reaction is not enough sensitive for detection, but the material is suitable for the usually less successful virus isolation.\n\nIt is a great result that the virus isolation attempts were successful with the GPAdV positive materials. It is more pity that the very interesting full genome sequence of these strains are not available. In the case of isolated viruses, it is only a small step by NGS. Because of the lack of full genome sequences of the examined AdVs, Figure 5. loses its relevancy. This makes some data incomparable in the figure.\n\nThe phylogenetic calculations are very interesting, however, the one, based on the full hexon gene, seems to show a bit different topology than the other two. It is surprising, that among mastadenoviruses, not the rodent AdVs are the most ancient ones as usual, and they are not monophyletic with the GPAdVs.\n\nSummarizing, this is a well-written manuscript with very relevant and precious results.\n\nMinor notes:\n\nThere are a couple of not disturbing misspellings or inadvertent mistakes. The only one, which can be confusing is in the legend of Figure 5. The labeling is correct on the figure, but in the legend, both columns are labeled with blue instead of orange and blue. Other mistakes:\n\nMisspelling in the abstract/background: “variable” instead of “variable”.\n\nTwice, the word “resuspended” was misspelled to “resuspened” (Methods/Immortalization, 1st paragraph). Also, here in the last sentence the word “aliquoted” was misspelled to “alliquoted”.\n\nIn most of the times, it was used correctly, but several times the “u” character was used instead of “µ” e.g.: twice in Methods/Immortalization, 2nd paragraph. Also, here a space is missing in one of the cases, between the number and the unit (1ug), correctly (1 µg). The lack of space mistake between the amount and the unit occurs several times in the manuscript. I won't detail all of them here. Please check them. Also, a dot is unnecessary in the middle of a sentence of this paragraph, after a catalog nr: (catalog # MF10096).\n\nA comma should be used in the listing instead of “and” at Methods/GPTEC-T cell line authentication, after (DSMZ ACC 057).\n\nThe shortening of fowl adenovirus D was used incorrectly. The letter “a” of adenovirus is in lower case font. It should be a capital “A”. Everywhere else it was used correctly. (Methods/Assembly, 1st paragraph: “FadV-D” was written instead of “FAdV-D”).\n\nOnce, the word “adenovirus” was written also with a capital incorrectly in the case of cynomolgus adenovirus.\n\nA “3’” end is missing for primer GPhexFW3 in Table 1.\n\nThe word “cytopathic” was misspelled to “cythopathyc” (legend of Figure 3.).\n\nThe word “twenty” was misspelled to “twety” (legend of Figure 4.). Also, here the word “absence” was misspelled to “absence”.\n\nThe use of abbreviation AdV species with the number of type like HAdV-C5 is not exactly appropriate, but widespread, so it is okay. But once it was misspelled (Methods/Assembly, 2nd paragraph: “HAdVA-12” instead of “HAdV-A12”). Also here in the whole paragraph, while in abbreviation the first letter is indeed in capital like “HAdV-A”, in writing the names of viruses should be written by lower case like “human adenovirus A12” according to the recommendation of the ICTV (if the name is from a Latin genus/species name, like Odocoileus hemionus adenovirus, it is correct with capital).\n\nThe name of fowl adenovirus in Methods/Assembly and in the legend of Figure 4 is incorrect (species D Fowl aviadenovirus 764). The correct name should be something like: “fowl adenovirus 8b strain 764 (FAdV-D)”.\n\nI would prefer the use of AdV type names (Arabic numbers) instead of species names (letters) on the phylogenetic trees, and GenBank numbers instead of RefSeq identification numbers (like starting with NC_...). These latter data won’t be updated, while the GenBank entries may be. Based on this, type data (including AC_... data) are more appropriate here.\n\nThe examined AdVs are originated from rodent hosts. I missed very much other available AdVs with rodent origin on the phylogenetic trees, like squirrel AdV (KY427939), or murine adenovirus 2 (HM049560). If the data was too much for the calculation, I would skip rather MAdV-1 or MAdV-3. They are more similar to each other, while MAdV-2 is more different.\n\nMaybe this is a new practice, but for me, it was surprising and indistinctive, that the manuscript referred to itself.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5283",
"date": "16 Mar 2020",
"name": "Adriana Kajon",
"role": "Author Response",
"response": "We would like to thank Dr. Vidovsky for his comments and suggestions for improvement of our paper. Below are our responses to each individual comment/suggestion: This manuscript describes the isolation of two strains of guinea pig adenoviruses and their successful propagation on a newly developed and on three commercially available guinea pig (GP) cell lines. The manuscript is very relevant, as every successful, novel virus isolation is very important for adenovirology. The manuscript also discusses the partial phylogenetic comparison of adenoviral sequences with the examined AdV strains (full hexon gene and partial polymerase and penton base sequence parts). The Introduction is thorough enough and dense in a good meaning. It only deals with guinea pig AdVs, but all the known publications are well collected. -The Methods section is a bit too detailed and long compared to the other parts of the manuscript. Sometimes the process is already published somewhere, and the manuscript refers to it correctly but also details shortly. This is unnecessary in some cases in my opinion. Response: The detailed description of methods and reagents is an F1000 Research requirement. -The process of the primary cell line production and its immortalization is very well-written. It is easy to follow the process and very interesting. The successful isolation and propagation of the two GPAdVs are a very nice job and well written. The pictures of CPEs on different cell lines represent the changes correctly and well visualize this part of the research. The surprising parts of the adenoviral PCR detection in the homogenates and infected cell cultures are that the original nasal scraping sample was PCR negative for AdV, while the cell culture infected by this material showed CPE and it was PCR positive. I would be curious of the opinion of the authors, how this can be possible, i.e., that the PCR reaction is not enough sensitive for detection, but the material is suitable for the usually less successful virus isolation. Response: We actually interpreted this result as a strong indicator that the original nasal scrapings sample contained infectious virus and that cell cultures were permissive for viral replication and amplification of the original input virus. -It is a great result that the virus isolation attempts were successful with the GPAdV positive materials. It is more pity that the very interesting full genome sequence of these strains are not available. In the case of isolated viruses, it is only a small step by NGS. Because of the lack of full genome sequences of the examined AdVs, Figure 5 loses its relevancy. This makes some data incomparable in the figure. The phylogenetic calculations are very interesting, however, the one, based on the full hexon gene, seems to show a bit different topology than the other two. It is surprising, that among mastadenoviruses, not the rodent AdVs are the most ancient ones as usual, and they are not monophyletic with the GPAdVs. Summarizing, this is a well-written manuscript with very relevant and precious results. Response: Since the submission of this manuscript, we have been working on obtaining whole genome sequences for GPAdV and expect to generate publications in the near future reporting those results. Minor notes: -There are a couple of not disturbing misspellings or inadvertent mistakes. The only one, which can be confusing is in the legend of Figure 5. The labeling is correct on the figure, but in the legend, both columns are labeled with blue instead of orange and blue. Response: We have corrected all spelling mistakes/typos in the body of the manuscript and figure legends. Other mistakes: -Misspelling in the abstract/background: “variable” instead of “variable”. Twice, the word “resuspended” was misspelled to “resuspened” (Methods/Immortalization, 1 paragraph). Also, here in the last sentence the word “aliquoted” was misspelled to “alliquoted”. In most of the times, it was used correctly, but several times the “u” character was used instead of“µ” e.g.: twice in Methods/Immortalization, 2 paragraph. Also, here a space is missing in one of the cases, between the number and the unit (1ug), correctly (1 µg). The lack of space mistake between the amount and the unit occurs several times in the manuscript. I won't detail all of them here. Please check them. Also, a dot is unnecessary in the middle of a sentence of this paragraph, after a catalog nr: (catalog # MF10096). Response: All of these corrections were incorporated into the revised version of the manuscript. -A comma should be used in the listing instead of “and” at Methods/GPTEC-T cell line authentication, after (DSMZ ACC 057). Response: The comma was NOT inserted because cell line 104 C1 was NOT obtained from the DSMZ collection. The text was edited to read “... HeLa (DSMZ ACC 057) and 293 (DSMZ ACC 305), and cell line 104C1 (ATCC CRL-1405) were used as negative controls” -The shortening of fowl adenovirus D was used incorrectly. The letter “a” of adenovirus is in lower case font. It should be a capital “A”. Everywhere else it was used correctly. (Methods/Assembly, 1 paragraph: “FadV-D” was written instead of “FAdV-D”). Once, the word “adenovirus” was written also with a capital incorrectly in the case of cynomolgus adenovirus. A “3’” end is missing for primer GPhexFW3 in Table 1. The word “cytopathic” was misspelled to “cythopathyc” (legend of Figure 3.). The word “twenty” was misspelled to “twety” (legend of Figure 4.). Also, here the word “absence” was misspelled to “absense”. The use of abbreviation AdV species with the number of type like HAdV-C5 is not exactly appropriate, but widespread, so it is okay. But once it was misspelled (Methods/Assembly, 2 paragraph: “HAdVA-12” instead of “HAdV-A12”). Also here in the whole paragraph, while in abbreviation the first letter is indeed in capital like “HAdVA”, in writing the names of viruses should be written by lower case like “human adenovirus A12” according to the recommendation of the ICTV (if the name is from a Latin genus/species name, like Odocoileus hemionus adenovirus, it is correct with capital). The name of fowl adenovirus in Methods/Assembly and in the legend of Figure 4 is incorrect (species D Fowl aviadenovirus 764). The correct name should be something like: “fowl adenovirus 8b strain 764 (FAdV-D)”. Response: All of these corrections were incorporated into the revised version of the manuscript. -I would prefer the use of AdV type names (Arabic numbers) instead of species names (letters) on the phylogenetic trees, and GenBank numbers instead of RefSeq identification numbers (like starting with NC_...). These latter data won’t be updated, while the GenBank entries may be. Based on this, type data (including AC_... data) are more appropriate here. Response: The original designations NC_034834 and NC_000942, were changed to KY306667 and AC_000012, respectively as requested in the Methods section and in revised Figures 4 and 5. -The examined AdVs are originated from rodent hosts. I missed very much other available AdVs with rodent origin on the phylogenetic trees, like squirrel AdV (KY427939), or murine adenovirus 2 (HM049560). If the data was too much for the calculation, I would skip rather MAdV-1 or MAdV-3. They are more similar to each other, while MAdV-2 is more different. Response: We completely agree with Dr. Vidovszky’s comments. We have made recent observations of close similarities between the genomes of GPAdV and MAdV-2 so the revisions addressing his comments are extremely important and timely. We have therefore incorporated the sequences for MAdV-2 into our new analyses for figures 4 and 5 and have revised both figures, and underlying data file now designated “GC Content values 030320.xls”."
}
]
},
{
"id": "58978",
"date": "19 Feb 2020",
"name": "Ana Paula Ravazzolo",
"expertise": [
"Reviewer Expertise Molecular virology",
"cell culture",
"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 manuscript, the authors described the generation of a tracheal epithelial guinea pig cell line, as well as the isolation and molecular characterization of two strains of adenovirus from Cavia porcellus (GPAdV). Virus propagation and partial nucleotide sequences are important findings, leading to the establishment of methods to better characterize GPAdVs.\nSome minor points: Title: Why using “initial propagation”? Why not “propagation” only?\nKeywords: Adding “phylogeny”, “nucleotide sequence” or “molecular characterization” could be more informative.\nMethods: In “Source of GP tracheas”, “…for transportation to the Kajon laboratory” do not specify the technical approach, like time and temperature until isolation and propagation of the cells. In “Immortalization of GP tracheal epithelial cells”, the sterile wash medium contains “2% penicillin/streptomycin”. Does it means 2 g of each one/100 mL of PBS? Alternatively, is it 2 times concentrated (2x)? The centrifugation will be better described in g than rpm. Why there are some additives to the culture medium (e.g. insulin, transferrin, etc.)? I suppose that they are used to select epithelial cells. If this is the case, some reference could be included. In PCR detection, it should be considered that positive controls are not mandatory to exclude false positives (as mentioned “To control for the specificity of the reaction in the absence of positive controls,…”)\nResults: Recovery of the virus in different cell lines seems to be similar for all of those evaluated although pictures showed at Fig. 3 correspond to different time lapses. Could we consider or speculate about more permissive cell lines? Legends of Figures: Fig 4 presents a misspelling – “twety” for twenty; in Fig. 5 blue is designed for both, virus genome and hexon gene.\nDiscussion: It is not clear why authors mentioned “apparent low efficiency” of virus propagation. This speculation was based on PCR? In comparison with other adenovirus? I suggest citing the reference or the reason to this statement.\nIn my opinion, there are some aspects to explore using the data obtained: differences of virus propagation among the cell lines, phylogeny, PCR positive result after virus propagation (nasal scraps) and comparisons with other described adenoviruses. One point that should be addressed is some low values of bootstrap and posterior probability at Fig. 4 to justify the conclusion “strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5284",
"date": "16 Mar 2020",
"name": "Adriana Kajon",
"role": "Author Response",
"response": "We would like to thank Dr. Ravazzolo for her comments on our manuscript. We have considered her suggestions and have introduced several edits as described below: -In this manuscript, the authors described the generation of a tracheal epithelial guinea pig cell line, as well as the isolation and molecular characterization of two strains of adenovirus from Cavia porcellus (GPAdV). Virus propagation and partial nucleotide sequences are important findings, leading to the establishment of methods to better characterize GPAdVs. Some minor points: Title: Why using “initial propagation”? Why not “propagation” only? Response: We have introduced the term “initial” referring to the propagation of GPAdV in cell culture to denote that these are early efforts towards the development of a robust system that can host efficient viral replication for stock production and titration and to. Reflect our awareness that our cell culture systems may change dramatically in the future as we develop and test new GP cell lines or implement the use of certain inhibitors (such Ruxolitinib, a Jak 1 and Jak 2 signaling pathway inhibitor) that decrease levels of inflammatory cytokines potentially making the system more permissive to infection. -Keywords: Adding “phylogeny”, “nucleotide sequence” or “molecular characterization” could be more informative. Response: We have added “phylogeny” and “nucleotide sequence” to the original keyword list in the revised version of our manuscript. Methods: -In “Source of GP tracheas”, “…for transportation to the Kajon laboratory” do not specify the technical approach, like time and temperature until isolation and propagation of the cells. Response: The text was edited to clarify this aspect of the protocol and read “ The tracheas were dissected at LRRI’s necropsy core and collected in 1X phosphate-buffered saline (PBS) for transportation to the Kajon laboratory in a refrigerated container within an hour” -In “Immortalization of GP tracheal epithelial cells”, the sterile wash medium contains “2% penicillin/streptomycin”. Does it means 2 g of each one/100 mL of PBS? Alternatively, is it 2 times concentrated (2x)? Response: Dr. Ravvazolo is absolutely right. This was an unnoticed typo. We have corrected the text to read “2X penicillin/streptomycin”. -The centrifugation will be better described in g than rpm. Response: The text in the Methods section was edited to read “800 rpm (150 RCF)…” -Why there are some additives to the culture medium (e.g. insulin, transferrin, etc.)? I suppose that they are used to select epithelial cells. If this is the case, some reference could be included. Response: These are standard protocols and medium formulations for primary cell culture so we have not provided any specific references. We did however edit the text to read “primary cell growth medium”. -In PCR detection, it should be considered that positive controls are not mandatory to exclude false positives (as mentioned “To control for the specificity of the reaction in the absence of positive controls,…”) Response: As written in the text, we DID NOT include positive controls. What we did was to submit amplicons for Sanger sequencing. Results: -Recovery of the virus in different cell lines seems to be similar for all of those evaluated although pictures showed at Fig. 3 correspond to different time lapses. Could we consider or speculate about more permissive cell lines? Response: We do not feel comfortable interpreting these results until we have a standardized system to titer input virus and monitor viral replication in culture. -Legends of Figures: Fig 4 presents a misspelling – “twety” for twenty; in Fig. 5 blue is designed for both virus genome and hexon gene. Response: These corrections were incorporated into the revised version of the manuscript. Discussion: -It is not clear why authors mentioned “apparent low efficiency” of virus propagation. This speculation was based on PCR? In comparison with other adenovirus? I suggest citing the reference or the reason to this statement. Response We do not have a reference to provide for this observation and we do not have quantitative PCR in place yet. Our statement is based on the observation of very limited cytopathic effect in subsequent passages. We have edited the text to address this issue and read “This study established the feasibility of propagating, albeit at an apparent low efficiency (based on the observation of modest cytopathic effect),…” -In my opinion, there are some aspects to explore using the data obtained: differences of virus propagation among the cell lines, phylogeny, PCR positive result after virus propagation (nasal scraps) and comparisons with other described adenoviruses. One point that should be addressed is some low values of bootstrap and posterior probability at Fig. 4 to justify the conclusion “strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus”. Response: We edited the text eliminating the word “strong\" to read “… in providing evidence confirming GPAdV as a unique species in the genus Mastadenovirus”."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1597
|
https://f1000research.com/articles/9-191/v1
|
16 Mar 20
|
{
"type": "Brief Report",
"title": "Children and adolescents on anti-retroviral therapy in Bulawayo, Zimbabwe: How many are virally suppressed by month six?",
"authors": [
"Silungile Moyo",
"Ronald Thulani Ncube",
"Hemant Deepak Shewade",
"Solwayo Ngwenya",
"Wedu Ndebele",
"Kudakwashe Collin Takarinda",
"Janet Dzangare",
"Tafadzwa Priscilla Goverwa-Sibanda",
"Tsitsi Apollo",
"Ronald Thulani Ncube",
"Solwayo Ngwenya",
"Wedu Ndebele",
"Kudakwashe Collin Takarinda",
"Janet Dzangare",
"Tafadzwa Priscilla Goverwa-Sibanda",
"Tsitsi Apollo"
],
"abstract": "Background: Zimbabwe is one of the countries in sub-Saharan Africa disproportionately affected by human immunodeficiency virus. In the “treat all” era, we assessed the gaps in routine viral load (VL) monitoring at six months for children (0-9 years) and adolescents (10-19 years) newly initiated on anti-retroviral therapy (ART) from January 2017 to September 2018 at a large tertiary hospital in Bulawayo. Methods: In this cohort study using secondary data, we considered first VL done within six to nine months of starting therapy as ‘undergoing VL test at six months’. We classified repeat VL≥1000 copies/ml despite enhanced adherence counselling as virally unsuppressed. Results: Of 295 patients initiated on ART, 196 (66%) were children and 99 (34%) adolescents. A total 244 (83%) underwent VL test at six months, with 161 (54%) virally suppressed, 52 (18%) unsuppressed and 82 (28%) with unknown status (due to losses in the cascade). Switch to second line was seen in 35% (18/52). When compared to children, adolescents were less likely to undergo a VL test at six months (73% versus 88%, p=0.002) and more likely to have an unknown VL status (40% versus 22%, p=0.001). Conclusion: At six months of ART, viral suppression was low and losses in the cascade high.",
"keywords": [
"Children living with HIV",
"Adolescent living with HIV",
"EAC",
"Mpilo",
"Operational research",
"SORT IT"
],
"content": "Introduction\n\nIn 2014, the Joint United Nations Programme HIV/AIDS (UNAIDS) announced ambitious new global 90-90-90 fast-track HIV targets for 20201. These targets were further supported by the 2016 “treat-all” WHO recommendations2. With the expansion of anti-retroviral treatment (ART) coverage, investments in the global response are shifting towards sustained viral suppression for improved survival and epidemic control. This is in the context of scaling up viral load (VL) monitoring to ensure 90% of people in care are virally suppressed (VL<1000 copies per ml)3. Globally in 2018, only 918 000 (54%) children aged 0–14 years living with HIV received ART4. HIV is among the top 10 leading causes of death among adolescents, a period where sustained adherence is particularly challenging, the only age group where deaths from HIV has not decreased5.\n\nZimbabwe is disproportionately affected by HIV. In 2017, 1.4 million people were living with HIV, with 5.8% being children 0–14 years6. An estimated 15% on ART have high VL7. The national ART guidelines recommend routine VL monitoring at six and 12 months, and then annually if stable on ART8. The extent to which routine VL monitoring is being implemented specifically for children and adolescents in the “treat-all” era has not been explored in Zimbabwe. We therefore assessed the gaps in routine VL monitoring at six months for children (0–9 years) and adolescents (10–19 years) initiated on ART at a large tertiary hospital in Bulawayo, Zimbabwe.\n\n\nMethods\n\nWe conducted a cohort study involving secondary data.\n\nMpilo Opportunistic Infections (OI) Clinic is within Mpilo Central Hospital in Bulawayo (the second largest city in Zimbabwe). It is a tertiary facility, managing complicated referrals, including patients on second and third line treatment. VL testing is offered as per national guidelines at the hospital HIV laboratory, adjacent to the clinic. Patient data are routinely entered in the electronic point of care database, ART register and patient care booklet8.\n\nFirst line ART regimen for children <3 years is ABC+3TC+LPV/R or AZT+3TC+LPV/r. Children 3–9 years and adolescents <35kg receive AZT+3TC+NVP or ABC+3TC+EFV, while adolescents >35kg receive TDF+3TC+NVP or EFV8. Those with VL ≥1000 copies/ml at six-months are offered enhanced adherence counseling (EAC), and a second VL test after three months. A repeat VL ≥1000 copies/ml (despite EAC) is classified as virally unsuppressed, and eligible for second line ART8.\n\nWe included all children (0–9 years) and adolescents (10–19 years) newly initiated on first line ART at Mpilo OI Clinic between January 2017 and September 2018.\n\nWe extracted anonymized patient data from electronic patient and laboratory databases, analyzed using STATA (version12.1 STATA Corp., College Station, TX, USA). If data were missing in electronic databases, we referred to paper-based registers and booklets. We defined low CD4 count as follows: CD4 count ≤350 cells/mm3 for children and adolescents >5 years, and CD4% <25% of total lymphocytes for children <5years8. We defined ‘undergoing VL testing at six months’ as those with first VL tests done within six to nine months of starting ART. Comparisons were made between children and adolescents using chi squared test.\n\nWe received ethics approval from Medical Research Council of Zimbabwe (MRCZ E/248), The Union Ethics Advisory Group, Paris, France (EAG 52/19) and the Mpilo Hospital Ethics Board. As the study involved review of secondary data, the ethics committee(s) waived the need for written informed consent.\n\n\nResults\n\nOf 295 patients initiated on ART during the study period, 196 (66%) were children and 99 (34%) adolescents. A total 141 (48%) were boys, 209 (71%) were WHO stage I or II, and 119 (40%) had severe anemia. Baseline CD4 count was available for 188, among whom 94 (50%) had low CD4 cell count.\n\nOf 295, a total of 244 (83%) underwent VL test at six months, which was significantly lower among adolescents when compared to children (73% versus 88%, p=0.002) (Figure 1). Of 295, 52 (18%) were virally unsuppressed, 161 (54%) virally suppressed and 82 (28%) unknown (unknown due to loss to follow up after ART initiation). Unsuppressed VL was not different among children and adolescents, though unknown VL suppression status, was higher among adolescents (40% versus 22%, p=0.001) (Table 1). Switch to second line was among 18 out of 52 eligible (35%), with no significant difference between adolescents and children (21% versus 39%, p=0.376) (Figure 1).\n\nCol%\n\nHIV=human immunodeficiency virus; ART=antiretroviral therapy.\n\n*repeat VL≥1000 copies per ml despite enhanced adherence counseling; **VL<1000 at six months or after repeat VL testing (post enhanced adherence counseling); ***not fitting into any of the above two categories, represents children or adolescents that were lost at any point in the cascade.\n\nHIV=human immunodeficiency virus; ART=antiretroviral therapy; VL=viral load; EAC= enhanced adherence counselling.\n\n\nDiscussion\n\nThis is one of the first studies from Zimbabwe attempting to assess the extent to which routine VL monitoring at six months is being implemented, specifically for children and adolescents in the “treat-all” era.\n\nOverall, one in five of those initiated on ART were virally unsuppressed at six months. The true estimate could be higher considering viral suppression was unknown for one-third of children. High unsuppressed VL and the observed attrition along the care cascade, undermines the last ‘90’ of the UNAIDS 90-90-90 targets in this special sub-population1. In this study, adolescents were more likely to be lost in the cascade compared to children, calling for focussed interventions for this sub-group. This is inspite of a comprehensive adolescent ART program at the clinic, and suggests a reversal of gains made earlier in the progarm9.\n\nCompared to findings in Harare, where two-thirds of those virally unsuppressed were switched to second line ART, only one-third were switched in our study10. Adherence to national ART guidelines should be an important priority focus in routine clinical mentorship11.\n\nFour in five underwent VL test by six months and EAC in our study, consistent with findings in Harare10. However, we found high proportion with VL≥1000 copies/ml at first and repeat testing. In Swaziland, children and adolescents were more likely to have high VLs and the least likely to achieve viral suppression. This calls for ART treatment support to address adherence problems of children and adolescents12.\n\nThe study had some limitations. Missing last visit dates prevented computation of proportion undergoing VL test among those retained at six-months. Missing baseline characteristics precluded analysis of factors associated with not undergoing a VL test and unsuppressed VL.\n\nIn conclusion, our study points to gaps in VL monitoring among children and adolescents in Bulawayo. Future studies are needed to understand reasons for attrition along the care cascade to better target interventions.\n\n\nData availability\n\nFigshare: Dataset for Moyo S et al. study: https://doi.org/10.6084/m9.figshare.c.4884726.v113\n\nFile ‘silungiledata’ contains all de-identified variables extracted for this study, alongside a codebook explaining all fields and field values.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe acknowledge Anna Fambira for her support in data extraction.\n\n\nDisclaimer\n\nThe views represented here are those of the authors and do not represent the institutions they are affiliated to.\n\n\nReferences\n\nUNAIDS: 90-90-90: A transformative agenda to leave no one behind. Geneva, Switzerland, 2014. Reference Source\n\nWorld Health Organization: Consolidated guidelines on the use of Antiretroviral drugs for treating and preventing HIV infection; 2016 recommendations for a public health approach. 2nd ed. Geneva, Switzerland. 2016. Reference Source\n\nPiot P, Quinn TC: Response to the AIDS pandemic--a global health model. N Engl J Med. 2013; 368(23): 2210–2218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUNAIDS: Fact Sheet - Global AIDS Update 2019. Geneva, Switzerland. Reference Source\n\nKim SH, Gerver SM, Fidler S, et al.: Adherence to antiretroviral therapy in adolescents living with HIV: Systematic review and meta-analysis. AIDS. 2014; 28(13): 1945–1956. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimbabwe Ministry of Health and Child Care: Zimbabwe National and Sub-National HIV estimates report 2017. Harare, Zimbabwe. 2018. Reference Source\n\nMinstry of Health and Child Care: Zimbabwe Population-based HIV Impact Assessment Report. Harare, Zimbabwe. 2016. Reference Source\n\nMinstry of Health and Child Care: Guidelines for Antiretroviral Therapy for the Prevention and Treatment of Guidelines for Antiretroviral Therapy for the Prevention and Treatment of HIV in Zimbabwe. Harare, Zimbabwe. 2016. Reference Source\n\nShroufi A, Gunguwo H, Dixon M, et al.: HIV-infected adolescents in southern Africa can achieve good treatment outcomes: results from a retrospective cohort study. AIDS. 2013; 27(12): 1971–1978. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBvochora T, Satyanarayana S, Takarinda CK, et al.: Enhanced adherence counselling and viral load suppression in HIV seropositive patients with an initial high viral load in Harare, Zimbabwe: Operational issues. PLoS One. 2019; 14(2): e0211326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: WHO recommendations for clinical mentoring to support scale-up of HIV care, antiretroviral therapy and prevention in resource-constrained settings. Geneva, Switzerland. 2005. Reference Source\n\nJobanputra K, Parker LA, Azih C, et al.: Factors associated with virological failure and suppression after enhanced adherence counselling, in children, adolescents and adults on antiretroviral therapy for HIV in Swaziland. PLoS One. 2015; 10(2): e0116144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShewade, HD: Codebook for dataset Moyo S et al study. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.11948475.v1"
}
|
[
{
"id": "61604",
"date": "08 Apr 2020",
"name": "Obinna Ikechukwu Ekwunife",
"expertise": [
"Reviewer Expertise Pharmacoeconomics",
"evidence-based public health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short report aimed to assess the gaps in routine VL monitoring at six months for children (0-9 years) and adolescents (10-19 years) newly initiated on anti-retroviral therapy from Jan 2017 to Sep 2018.\nThis study is essential as such data is needed to assess how programs are fairing with regards to the UNAIDS 90-90-90 target.\nThe study was succinctly reported. All the essentials results based on their study objective were addressed. The study should be accepted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5466",
"date": "04 May 2020",
"name": "Hemant Deepak Shewade",
"role": "Author Response",
"response": "Thank you for the constructive comments"
}
]
},
{
"id": "61601",
"date": "28 Apr 2020",
"name": "Brian Van Wyk",
"expertise": [
"Reviewer Expertise HIV/AIDS",
"Health Systems",
"Adolescent Health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study reports on viral load monitoring at 6 months for of children and adolescents who were initiated on HIV treatment in a tertiary hospital in Bulawayo. The study is important because of the HIV epidemic in Zimbabwe, and the need to reach the third 90 of UNAIDS 90-90-90 targets.\nThe methodology is sound and clearly reported on. Appropriate statistical analysis is done, and these are aligned with the objectives of the study.\nFew other sociodemographic and clinical factors were collected and analysed; which is a limitation to the study. This should be indicated.\n\nIn the discussion, enhanced adherence counseling is mentioned as being implemented in the hospital. However, little information on this is provided in the background. Also, it would be useful if the analysis could report on how many of the current cohort received enhanced adherence counseling.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5467",
"date": "04 May 2020",
"name": "Hemant Deepak Shewade",
"role": "Author Response",
"response": "Thank you very much for your constructive comments. ReviewerFew other sociodemographic and clinical factors were collected and analysed; which is a limitation to the study. AuthorsWe have included this and mentioned it as a limitation in the last but one paragraph of the discussion section. We hope this is fine. ReviewerIn the discussion, enhanced adherence counseling is mentioned as being implemented in the hospital. However, little information on this is provided in the background. Also, it would be useful if the analysis could report on how many of the current cohort received enhanced adherence counseling.AuthorsWe have information regarding when enhaned adherence conselling is provided in the settings section. We hope this is fine. We have provided information regarding how many recevied enhanced adherence counselling in this cohort. Please see figure 1.We hope our response is satisfactory and the current version does not require any edits. Hence, we are not submitting a revised manuscript."
}
]
},
{
"id": "61603",
"date": "26 May 2020",
"name": "Catherine Kegakilwe Koofhethile",
"expertise": [
"Reviewer Expertise Immunology",
"HIV"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that this manuscript is addressing a very important gap in knowledge that is relevant for the current ‘treat all’ recommendations. They accessed the gaps in routine viral load monitoring at six months for children and adolescents who initiated antiretroviral therapy in a hospital in Zimbabwe. Their sample number is good enough for this analysis. The manuscript is very well written, it is very clear and concise. The study was based on analysis of secondary data which was approved by IRB.\n\nI only have one comment that need clarification- the authors keep comparing their analysis with a study done in Harare and it is not clear whether this study that they are comparing to was conducted on adult population or the same population as they describe in their analysis. This needs to be clarified. In addition, they need to explain what could be accounting for the differences found.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-191
|
https://f1000research.com/articles/9-189/v1
|
16 Mar 20
|
{
"type": "Research Article",
"title": "Exploring the perceptions, practices and challenges of gestational diabetes detection and management among health care providers in a district of Bangladesh",
"authors": [
"Abul Halim",
"Abu Sayeed Md. Abdullah",
"Fazlur Rahman",
"Animesh Biswas",
"Abu Sayeed Md. Abdullah",
"Fazlur Rahman"
],
"abstract": "Background: Gestational diabetes mellitus (GDM) is one of key maternal morbidities during pregnancy globally. It is one of the neglected threats during pregnancy that constitutes pregnancy-related complications. Moreover, a significant number of GDM positive women later develop type 2 diabetes after delivery following an interval of 5-10 years. However, the real magnitude of GDM in Bangladesh is still undetermined. The objective of this study was to explore the perceptions, practices and challenges of GDM detection and management among health care providers (HCPs) in the Tangail district of Bangladesh. Methods: A mixed method study was conducted between November 2017 and January 2018. Quantitative data was collected through a survey of providers, whereas qualitative data collection included in-depth interviews with providers and direct observation of 41 health care facilities. Descriptive analysis was performed for quantitative data and qualitative data analysis was performed thematically. Results: The study revealed that 66.6% of HCPs did not know about GDM related complications for affected mothers and their babies, 81.2% did not know about the risk factors of GDM, 95.8% were not aware about the management and/or treatment of GDM, 79.1% never conducted GDM screening of pregnant women and almost none of the interviewees received any special training on GDM. We observed that only 31.3% of women were tested for blood sugar in antenatal care and of those, 12.5% were identified as having elevated blood sugar levels. GDM related information was collected in 4.2% of cases. Conclusions: There is limited knowledge and skills among the HCPs to detect and manage GDM at the community level. More programmatic emphasis is required to strengthen the capacity of community health care workers in detecting and treating GDM.",
"keywords": [
"Gestational diabetes mellitus",
"community",
"perception",
"practice",
"challenges",
"Bangladesh"
],
"content": "Introduction\n\nDiabetes in pregnancy is a neglected cause of maternal morbidity, with one in every six pregnancies affected by GDM globally1. Around 88% of cases of GDM are found in low- and middle-income countries2. Prevalence of GDM varies from 1% to 28% depending on population characteristics, diagnostic measurement and screening methods, with the South East Asian region indicated to have a high prevalence (24.2%)3. Bangladesh is one of the top ten contributors to diabetes cases globally and this number is expected to double in the next 20 years4. It is estimated that in Bangladesh alone, more than two million people suffer from diabetes but are as of yet undiagnosed5. The current prevalence of diabetes is 5.5% and the expected rise to 8.2% by 2030 renders diabetes in Bangladesh a major public health problem that has to be addressed by the national health system in the near future4,6. Diabetes is associated with transitions in lifestyle and industrialization, which is more common in urban areas but increasingly extends to rural settings7.\n\nFindings in Bangladesh have shown that 23% of patients with diabetes mellitus are treated at outpatient departments of upazila health complexes (primary health care centers) and 23% in district hospitals (secondary health care centers)8. In the past five years, there has been an increased focus in Bangladesh on improving service delivery at the primary level and at referral centers, including union health and family welfare centers (the lowest level of primary health care centers), as well as at the community level through community clinics and community health workers9. Nevertheless, most of the programme still focuses on the provision of emergency obstetric and newborn care, with poor focus on other maternal and newborn health related areas that are on the incline, such as the impact of non-communicable diseases on maternal and newborn health10. A new operational plan provides a structure for improving care with regards to preconception, pregnancy, childbirth and the immediate postpartum period11. Areas prioritized for the provision of quality maternal and neonatal health services include populations with a high maternal mortality ratio and those living in impoverished areas and facing geographical and social disadvantages12. Bangladesh has developed the 2013–2020 Action Plan for the prevention and control of non-communicable diseases, a national strategy for maternal health that highlights the importance of GDM13.\n\nThe prevalence of GDM ranges from 8.2% in rural Bangladesh14 to 12.9%15, with estimates dependent on the diagnostic criteria used16. Undiagnosed women with diabetes in pregnancy are a key concern in Bangladesh due to the low social status of women, which results in inequalities in healthcare access and nutrition17. Women often do not receive treatment for sickness and therefore often resort to self-medication or seek care from unqualified practitioners18. Women with GDM have an increased risk of developing obstetric complications like prolonged labor and are prone to develop type-2 diabetes in the future. Their babies might be born overweight and develop childhood obesity or type-2 diabetes later in life19. As there is already an existing community network, this can be used to refer all pregnancies that have been identified at the community level for antenatal care (ANC) and GDM screening facilities20.\n\nThe aim of this study is to determine the perceptions and practices of GDM detection and management in a selected sub-district of Bangladesh and explore existing barriers to adequate detection and management of GDM.\n\n\nMethods\n\nA total of 47 face-to-face surveys and 10 in-depth interviews (IDIs) were conducted with health care providers (HCPs) and with health managers. Furthermore, 30 ANC consultations were observed at 30 primary facilities at the community, union health center and upazila health complex level. Finally, 41 facility inventories were carried out using a preformatted checklist. Table 1 provides a summary of the study methods.\n\nThis was a mixed methods study applying both quantitative and qualitative methods. It was conducted from November 2017 to January 2018 in the Mirzapur sub-district of Tangail district, Bangladesh. The sub-district has a population of 274,088, with 65 facilities including 53 community clinics, 10 union health and family welfare centres, one upazila health complex and one medical college hospital (tertiary facility). Out of 63 primary health care facilities (community clinics and union health and family welfare centers), 30 community clinics and nine union health and family welfare centers were randomly selected by simple randomization, achieved via the random number generator in Microsoft Excel 2010. The study also included the upazila health complex and medical college hospital for data collection.\n\nA structured questionnaire was developed by the research team of the study, available as Extended data21. This questionnaire was pretested outside the study area to finalize the questionnaire. Pretesting was carried out among HCPs at one community clinic and one union health and family welfare centre in a different upazila within the district of Tangail. No changes were made to the questionnaire after pre-testing and no validity testing was performed. Then, the structured questionnaire was used to collect quantitative data from selected HCPs, which include grassroots level health workers, such as health assistants (HAs), family welfare assistants (FWAs), health inspectors (HIs), family planning inspectors (FPIs), and community health care providers (CHCPs), and also health workers from the lowest level primary health care centers such as family welfare visitors (FWVs) and sub-assistant community medical officers (SACMOs) (Table 1). The questionnaire included general information, health workers’ perspectives on GDM and availability of equipment and logistics to provide services for GDM patients in the primary health care centers. An official letter from the Mirzapur Upazila Health Complex was circulated among the health workers (n=52) in the 41 facilities, which explained the objectives and invited them to participate in the study. A copy of this letter is available as Extended data21. Four trained field data collectors were responsible for collecting data. Health workers were invited and those who willingly wanted to participate in the study were enrolled purposively. Five of the invited participants were not willing to participate as they were busy at that time with their own work. Data collection was conducted at their place of work and it took approximately 20 minutes to complete each of the face to face interviews.\n\nTwo research assistants (anthropologists) were assigned to collect qualitative data through IDIs with the HCPs working at the facility level. A pretested interview guide was used for the interviews, available as Extended data21. Pretesting was carried out among HCPs at one community clinic and one union health and family welfare centre in a different upazila within the district of Tangail. Minor changes were made to the interview guide after pre-testing. No validity testing was performed. HCPs were chosen purposively and written consent was sought before participation in the face-to-face interviews. Out of 13 respondents, three participants weren’t able to participate in the IDIs due to their other engagements. The research assistant described the objectives of the study before data collection. Each of the interviews took approximately 20–25 minutes and were conducted at the facilities. Interviews were audio recorded upon permission and the research assistant took some key notes.\n\nA total 30 ANC checkups were observed at different facility levels. Of them, eight were at Kumudini hospital, two at the upazila health complex, eight from union health and family welfare centers and twelve from community clinics. Four trained field data collectors used a pretested checklist to do the observations, available as Extended data21. Pretesting was carried out among HCPs at one community clinic and one union health and family welfare centre in a different upazila within the district of Tangail. No changes were made to the checklist after pre-testing and no validity testing was performed.\n\nPermission to collect the data was obtained from the health department, the patients and HCPs. The checklist contained some key areas including infrastructure, logistics, waste management and staff. The observation was made during routine antenatal check-ups at the ANC area of the facility by a doctor or nurse during the hospital working hours.\n\nFacility inventory was conducted for 31 purposively selected community clinics, eight union health and family planning centers, one upazila health complex and from the Kumudini hospital. The field level data collectors used a structured checklist to collect the inventory data, available as Extended data21. Key areas covered supplies included furniture and durable equipment, clinical supplies and other optional equipment. During this process, the data collectors used hospital records and sought support from the responsible persons to fill out the checklist.\n\nQuantitative data were cleaned and missing data were checked by the data supervisors. If any data were missing, the data collector revisited the home using previously collected sociodemographic data to contact participants for this purpose and collected the missing data from that responder. SPSS statistical software (version 21.0 for windows) was used for descriptive analysis.\n\nThe qualitative data were transcribed from the local language (Bengali) from audio recordings and translated into English by the anthropologists. The principal researcher randomly selected transcripts (10%) by simple randomization to review the quality of transcripts. Peer debriefing was also performed to maintain the reliability of the data. Initial open coding was performed and themes were identified, followed by selective coding, done manually by repeated reading of the transcripts. Themes were identified from the data22,23 and analyzed thematically.\n\nThe study is under the project named ‘Evaluating community-based detection and surveillance of gestational diabetes’, which has been approved by the national ethical review committee of Centre for Injury Prevention and Research, Bangladesh (CIPRB) (Memo - CIPRB/ERC/2017/20). Informed written consent was received from each of the interviewees for participation before collecting their data for the study.\n\n\nResults\n\nThe results section of this paper is presented in two sections. The first section contains information about the perceptions, knowledge, attitude and practices of the Health Care Providers (HCPs) and health managers on GDM through the provider survey and qualitative IDIs. The second section presents the findings of the ANC observations and the facility inventory checklists.\n\nBasic knowledge about GDM. The majority of the respondents who participated in the survey had never heard of diabetes that occurs during pregnancy or gestational diabetes (81%, n=38). Furthermore, 81.2% of respondents (n=38) were not aware of the risk factors for GDM and among them, 16.6% (n=8) stated that there are no risk factors (Figure 1). Data from the IDIs gave similar findings. HCPs had no clear perception of GDM and some considered GDM to be the same as diabetes.\n\n“There is no difference between the diabetes and gestational diabetes. When diabetes occurs in pregnancy then it is called gestational diabetes and will not cure like diabetes. All the management and treatment of diabetes during pregnancy is similar to the diabetes.” (P2, Community Health Care Provider (CHCP))\n\nKnowledge on the complications of GDM. Around 66.6% (n=31) of HCPs did not know about GDM related complications for mothers and their babies. Furthermore, 16.7% (N=8) thought that there are no GDM related complications and 16.7% (n=8) knew that there are some GDM related complications that could affect mothers and babies (Figure 2). Some respondents stated newborns could develop abnormally, pregnant mothers may have diabetes later in life and some might develop unspecific problems as a result of GDM complications.\n\n“I don’t know clearly about the complications. But I perceive that a GDM mother has more chances for eclampsia, pre-eclampsia, intra-uterine death, stillbirth, pre-term delivery.” (P7, HI)\n\nKnowledge on the GDM tests. Among the 47 respondents, 54.1% (n=25) said that a GDM screening test is essential for pregnant women, 29.1% (n=14) did not know and 16.6% (n=8) said a screening test is not necessary during pregnancy. Almost none (97.8%, n=46) could mention the exact timing for when a screening test should be performed during pregnancy (Figure 3). None of the participants (100%, n=47) had knowledge of the screening process.\n\n“We have no clear idea on gestational diabetes screening test of pregnant mothers. The test need to done during antenatal care; however, we do not practise. We need training on GDM and logistics for screening need to available during ANC.” (P4, CHCP)\n\nKnowledge about GDM treatment. Regarding treatment, 95.8% (n=45) of the respondents did not know if there was any treatment for GDM available and 4.1% (n=2) said there is no treatment. Regarding delivery, 4.1% (n=2) mentioned that GDM patients should deliver by caesarian section and 95.8% (n=45) had no idea about the recommended mode of delivery for a woman with GDM. Regarding the necessity for follow-up after delivery, 70.8% (n=33) did not know whether follow-up was necessary and 22.9% (n=11) said postpartum follow-up is not needed for women with gestational diabetes.\n\n“I think exercise and insulin are the only treatment for mothers who are suffering from diabetes during pregnancy” (P3, FPI)\n\nGDM screening. Regarding the screening practice, 79.1% (n=37) of HCPs never screened pregnant women for GDM and 18.7% (n=9) had no idea about screening. In addition, 68.7% (n=32) did not know what tests are available to screen for GDM in their health facility/department and 20.8% (n=10) said they have no screening equipment at their facility. One of the respondents described how to do a blood sugar test, but none of the other respondents knew the process (97.8%, n=46).\n\nGDM management practices. Almost all (95.8%, n=45) of the providers did not know about a management protocol for GDM affected mothers. Regarding advice given to GDM positive mothers, 91.6% of providers (n=43) did not know what to advise, 6.2% (n=3) said they do not give any advice and only one HCP (2.1%, n=1) recommended exercise, diet and insulin for GDM mothers. Almost all (95.7%, n=45) did not know about drug therapy for GDM mothers. Some (4.3%, n=2) perceived that both oral medicine and insulin can be used for GDM management.\n\n“We don’t have any management protocol for gestational diabetes. I perceive that after a woman is diagnosed, she is immediately referred to hospital for further treatment.” (P6, CHCP)\n\n“Insulin is the only drug of choice for a GDM patient” (P8, FWV)\n\n“In our hospital if a mother identified with diabetes during pregnancy, we refer to district hospital for management” (P-10, Nurse)\n\nFollow-up of GDM patients. Around half (56.2%, n=26) of respondents did not know about follow-up for GDM patients. Additionally, 27.1% (n=13) providers said they did not advise GDM mothers to go for a blood glucose checkup at secondary or tertiary facilities and 62.5% (n=29) did not know what to advise a mother suffering from GDM. Nearly all of the providers (97.9%, n=46) did not know about follow-up for women who had GDM after delivery. None of the respondents knew about newborn blood glucose checkups after delivery.\n\n“I advised the mothers having diabetes during pregnancy to do a blood glucose test on a weekly basis at the medical college hospital” (P2, CHCP)\n\nNearly none of the providers (97.9%, n=46) received any special training on diabetes mellitus and/or GDM during training or on the job. Many of the interviewed providers (97.9%, n=46) never got an opportunity to deal with GDM patients. Moreover, there are limited resources (equipment, material) for testing in their facilities. As the majority of providers are not very knowledgeable on GDM, they do not feel competent to discuss the topic with the women who come for ANC. In terms of equipment, 93.7% (n=44) of the providers said they have only blood pressure devices. Only two providers (4.2%) stated that glucometers are available in their facilities and one provider (2.1%) stated the availability of a blood pressure device, an ultrasound machine, urine dipstick and leaflets on nutrition at her facility.\n\n“I only heard about the term gestational diabetes. But I never received any training on screening. I even have a limited idea on counseling pregnant women to prevent GDM” (P1, FPI in IDI )\n\n“We don’t have any special facilities for GDM management at our union family welfare center.” (P10, FWV in IDI)\n\nA total of 30 ANC sessions were observed in the facilities. In 31.3% of observed consultations (n=9), blood sugar test related information was collected. Of all the observations, a high blood sugar level was found in 12.5% (n=4). In only one (4.2%) of the ANC observations did the patient receive GDM related information. None of the patients were asked about any previous history of GDM. In 45.8% (n=14) of the observations, the blood pressure of the pregnant woman was measured. In none of the ANC observations did providers counsel on GDM and provide information on the risk of developing diabetes later in life, the disappearance of GDM after pregnancy or when to diagnose. Furthermore, in none of the observed ANC consultations was a capillary sugar test or urine sugar test carried out, nor were any women referred to a laboratory for a sugar test (Table 2).\n\nKH, Kumudini Hospital; UHC, Upazila Health Complex; FWC, Family Welfare Centre; CC, community clinic; BMI, body mass index; GDM, gestational diabetes mellitus.\n\nThe inventories of the facilities were assessed using a structured checklist. Among the 41 facilities assessed, 95.1% (n=39) provided outpatient services. All facilities provided ANC and postnatal care (PNC) but limited equipment for essential tests like glucose tests, urine tests and hemoglobin tests for ANC. Only two (4.8%) facilities had a separate breast-feeding corner, ANC corner and PNC corner and equipment for ultrasonography and hemoglobin testing. The referral facility (Kumudini Hospital) had a consultant trained on GDM. Nine (21.9%) of the facilities provided 24/7services.\n\nRecord keeping. During September to November 2017, a total of 6805 pregnant women received ANC from 41 selected facilities. Only six GDM women were detected in the referral hospital, whereas other facilities didn’t identify any women. Among the 41 facilities, 78% (n=32) did not perform any deliveries in last three months. A total of 31 preeclampsia/eclampsia cases, 125 premature babies, 215 cases of premature membrane rupture, 45 still births and four maternal deaths were reported in Kumudini hospital but such records about obstetric and newborn complications were not available in the other facilities (Table 3).\n\nKH, Kumudini Hospital; UHC, Upazila Health Complex; FWC, Family Welfare Centre; CC, community clinic; GDM, gestational diabetes mellitus; CS, caesarean sections; ANC, antenatal care; NVD, normal vaginal deliveries; PROM, premature rupture of membranes.\n\nHuman Resources. Only 29.2% (n=12) of the facilities have an obstetric consultant and 17% (n=7) have a SACMO, none of whom were trained on diabetes. Moreover, 26.8% (n=11) have a midwife/FWV, almost all (93.5%, n=29) community clinics have CHCPs and 41.4% (n=17) facilities have community skilled birth attendants (CSBAs). GDM-trained medical officers and nurses are only available in Kumudini hospital.\n\nEquipment and resources. Weighing machines were present in all facilities, 26.8% (n=11) had glucometers but only 4.8% (n=2) had glucose test strips. In terms of other equipment, 95.1% (n=39) had blood pressure devices, 7.3 % (n=3) had a fetoscope, 12.1% (n=5) had an equipped labor room (with remaining facilities having an unequipped labor room), 87.8% (n=36) had a patient examination table and 95.1% (n=39) had a baby weighing machine (scale). Laboratory testing and the availability of ultrasound scanning existed only at Kumudini Hospital and Mirzapur Upazila Health Complex. Supplies of glucose to do oral glucose tolerance tests were available only at the Mirzapur Upazila Health Complex. Metformin tablets and insulin injections were not available at any of the facilities (Table 4).\n\nKH, Kumudini Hospital; UHC, Upazila Health Complex; FWC, Family Welfare Centre; CC, community clinic.\n\n\nDiscussion\n\nGestational diabetes is still undetermined in the rural communities of Bangladesh. The primary health care facilities are not well equipped for GDM screening. The perception of, knowledge of and practices regarding GDM among the HCPs were found to be very poor. The majority of the findings show that HCPs do not know about GDM. There is a serious gap between the knowledge of diabetes (any type) and diabetes during pregnancy. The capacity of HCPs and resources for GDM detection and management were not available in the facilities.\n\nAn assessment of the primary health care facilities in Bangladesh found limitations regarding resources and equipment for the diagnosis and treatment of diseases. The maternal health care services were also found to be poor, especially essential tests like glucose, hemoglobin and urine protein ANC tests. HCPs at the community level were found to be incapable of conducting all the tests on pregnant women9. This study also found similar results.\n\nThe maternal health task force in 2017 raised the issue of GDM as one of the important neglected causes of maternal mortality and morbidity. The diagnosis of women with GDM during ANC was found to be neglected among the HCPs2,3, which supported this study finding. The UN Sustainable Development Goals for 2030 also emphasized the prevention of non-communicable diseases, especially gestational diabetes3.\n\nCommunity health workers have already proved to be important in improving diabetes outcomes in low-income countries24. Besides this, community health workers are also useful in patient education, identification and referral of high-risk mothers25. In our study, the health workers did not have sufficient knowledge to deal with GDM. However, the well-structured health system in Bangladesh and experienced community health workers at the grassroots level are key strengths for improving the identification and treatment of GDM.\n\nUntil now, there has been no routine screening of all pregnant patients for gestational diabetes in Bangladesh. However, a national maternal and neonatal health guideline has been approved by the Ministry of Health and Family Welfare for the systematic management of affected patients based on current evidence and best practice.\n\nLo et al. conducted a qualitative study on the provider view on GDM and found a lack of depth of understanding and skills regarding GDM. A lack of knowledge and misperception were also found among the providers. Although specialist care is available at tertiary centers that are represented by medical colleges, GDM related activities conducted at health services, particularly at primary level, are erratic or not available26.\n\nA cross-sectional survey on the equipment available for GDM screening in low- and middle-income countries found that primary health care facilities at a community level were not well equipped for GDM screening and management. There was a lack of medicine for the treatment of GDM indicated in that study, which also found similar barriers and challenges for the facilities to the current study27. Another study in Morocco also described similar findings regarding the knowledge and skill gaps of HCPs on GDM28.\n\nThis study addressed the perception and practices of HCPs on GDM screening and management at upazila, union and community levels in Mirzapur, Tangail district. Existing barriers for adequate detection and management of GDM are also identified.\n\n\nConclusion\n\nGDM is one of the major public health issues in Bangladesh. There is a significant need for the development of knowledge and skills of HCPs to detect GDM in the community. Quality ANC during pregnancy is needed to ensure GDM screening for every pregnant woman to reduce the burden of and morbidity due to GDM in Bangladesh.\n\n\nData availability\n\nData is stored at the Centre for Injury Prevention and Research, Bangladesh (CIPRB). Due to sensitivity of the data (contains identifying information), permission is required from the Ethical Review Committee (ERC) of CIPRB, Dhaka, Bangladesh for sharing data with a third party. Data can be requested from the CIPRB, who will contact the ERC to gain approval to share the data. The conditions for gaining data access are a formal request with a clear objective and formal permission from the ERC. Please contact the corresponding author in order to request the data through email at info@ciprb.org.\n\nFigshare: Qualitative tools for GDM study in Bangladesh. https://doi.org/10.6084/m9.figshare.11925318.v121\n\n- Survey Questionnaire.docx\n\n- IDI_Discussion Guide.docx\n\n- Revised ANC observations checklist.docx\n\n- Inventory Checklist.docx\n\n- Copy of official letter.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe would like to acknowledge support of Dr Bettina Utz, PhD of Robert Koch Institut, Germany for her support and contribution in research development, analysis plan, review of the manuscript.\n\n\nReferences\n\nInternational Diabetes Federation: Gestational diabetes. In: Care & prevention Improving the quality of life of people with diabetes and those at risk. 2017. Reference Source\n\nIversen K: Diabetes in Pregnancy: A Neglected Cause of Maternal Mortality. In: Maternal Health Task force. 2017. Reference Source\n\nMuche AA, Olayemi OO, Gete YK: Prevalence and determinants of gestational diabetes mellitus in Africa based on the updated international diagnostic criteria: a systematic review and meta-analysis. Arch Public Health. 2019; 77: 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkter S, Rahman MM, Abe SK, et al.: Prevalence of diabetes and prediabetes and their risk factors among Bangladeshi adults: a nationwide survey. Bull World Health Organ. 2014; 92(3): 204–213A. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNordisk N: Changing diabetes in Bangladesh through sustainable partnerships. Bluepr Chang Program. 2012; 4: 1–24. Reference Source\n\nWorld Health Organization: Global Report on Diabetes. Isbn. 2016; 978: 88. Reference Source\n\nRamachandran A, Snehalatha C: Prevention of diabetes: How far have we gone? Indian J Endocrinol Metab. 2014; 18(3): 252–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUSAID: Bangladesh: Standard DHS, 2011. DHS Progr. 2011. Reference Source\n\nSohail M, Pritchard AP, Weekly P, et al.: Accessibility and quality of government primary health care: achievement and constraints. Bangladesh Dev Stud. 2005; 31(3): 63–98.\n\nTorpey K, Mastro TD: Discussion paper on how to promote the inclusion of the prevention and control of noncommunicable diseases within other programmatic areas. 2016; 1–25. Reference Source\n\nDean SV, Lassi ZS, Imam AM, et al.: Preconception care: Promoting reproductive planning. Reprod Health. 2014; 11 Suppl 3: S2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUSAID: Acting on the call: Ending preventable child and maternal deaths. US Agency Int Dev New York. 2016; 1–104. Reference Source\n\nBegum IA: Gestational Diabetes Mellitus in Primi Gravida of Bangladesh in Different Trimesters. Int J Biol. 2014; 6(3): 18–23. Publisher Full Text\n\nSayeed MA, Mahtab H, Khanam PA, et al.: Diabetes and hypertension in pregnancy in a rural community of Bangladesh: a population-based study. Diabet Med. 2005; 22(9): 1267–71. PubMed Abstract | Publisher Full Text\n\nJesmin S, Akter S, Akashi H, et al.: Screening for gestational diabetes mellitus and its prevalence in Bangladesh. Diabetes Res Clin Pract. 2014; 103(1): 57–62. PubMed Abstract | Publisher Full Text\n\nCarolan M: Gestational diabetes mellitus among women born in South East Asia: a review of the evidence. Midwifery. 2013; 29(9): 1019–26. PubMed Abstract | Publisher Full Text\n\nHannan C: Moving forward on women, gender equality and diabetes Presentation by Carolyn Hannan. 2008; 11. Reference Source\n\nAhmed SM, Adams AM, Chowdhury M, et al.: Changing health-seeking behaviour in Matlab, Bangladesh: do development interventions matter? Health Policy Plan. 2003; 18(3): 306–15. PubMed Abstract | Publisher Full Text\n\nRahman M, Tahmin T, Ferdousi S, et al.: Gestational Diabetes Mellitus (GDM): Current concept and a short Review. Bangladesh J Pathol. 2009; 24(1): 16–20. Publisher Full Text\n\nWorld Health Organization (WHO): WHO Recommendation on Antenatal care for positive pregnancy experience. WHO Recomm Antenatal care Posit pregnancy Exp. 2016; 152. Reference Source\n\nHalim A, Abdullah AS, Rahman F, et al.: Qualitative tools for GDM study in Bangladesh. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11925318.v1\n\nIrving S: Interviewing as Qualitative Research - A Guide for Researchers in Education and the Social Sciences. Teach Coll Columbia Univ USA. 2006. Reference Source\n\nBoyatzis RE: Transforming qualitative information: Thematic analysis and code development. Sage. 1998. Reference Source\n\nPerry HB, Zulliger R, Rogers MM: Community health workers in low-, middle-, and high-income countries: an overview of their history, recent evolution, and current effectiveness. Annu Rev Public Health. 2014; 35(1): 399–421. PubMed Abstract | Publisher Full Text\n\nAlaofè H, Asaolu I, Ehiri J, et al.: Community Health Workers in Diabetes Prevention and Management in Developing Countries. Ann Glob Health. 2017; 83(3–4): 661–675. PubMed Abstract | Publisher Full Text\n\nLo C, Ilic D, Teede H, et al.: Primary and tertiary health professionals' views on the health-care of patients with co-morbid diabetes and chronic kidney disease - a qualitative study. BMC Nephrol. 2016; 17(1): 50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUtz B, De Brouwere V: \"Why screen if we cannot follow-up and manage?\" Challenges for gestational diabetes screening and management in low and lower-middle income countries: results of a cross-sectional survey. BMC Pregnancy Childbirth. 2016; 16(1): 341. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUtz B, Assarag B, Smekens T, et al.: Detection and initial management of gestational diabetes through primary health care services in Morocco: An effectiveness-implementation trial. PLoS One. 2018; 13(12): e0209322. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "127221",
"date": "31 Mar 2022",
"name": "John Punnose",
"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 study gives insight into inadequacies of GDM awareness, GDM monitoring and management in Bangladesh. It may be considered as a pilot study, as there are major concerns to select it as a full scientific paper. Main objections are the validity of the conclusions based on the limited data. No statistical analysis. The presentation of data requires substantial modifications. Please refer to my comments to authors.\nComments to authors: This study is an attempt to identify the GDM knowledge among Health Care Personnel, assess the infrastructure of the health care facilities and to audit the present GDM screening strategies in a selected sub-district of Bangladesh. The authors have designed an acceptable study protocol (without prior validation) and applied it to selected health centers. The study conveys suboptimal/highly inadequate GDM management facilities/skills at community level in Bangladesh.\nAs a pilot study, the findings of this study is useful to plan augmentation of GDM screening facilities and management skills of health care workers in Bangladesh and other developing countries. But as a scientific study, there several concerns for this study.\nWas a proper sample size calculation done for selecting the number of centers and health care workers? The detailed interviews were limited to 10 participants. Is the number enough to draw conclusions of national significance (for Bangladesh)?\n\nIntroduction 3rd paragraph: \"Increased risk of obstetric complications like pronged labor\". Many more important complications are missed.\n\nIntroduction last paragraph; \"...explore existing barriers to adequate detection and management of GDM\". This is put as an aim, but there are no attempts in the study to find the barriers in the study.\n\nThe methodology especially selection of study population is presented in a confusing manner. I suggest a diagram showing different arms, showing the selection of centers, ANC checks, facility checking etc. Table 1 gives this data in a different form but using titles like Qualitative and Quantitative as the main heading confuses the reader.\n\nThe presentation of findings needs to be improved e.g. is Fig. 1 with yes/no pie diagram required for a study like this? The authors can state the finding in the manuscript.\n\nFig. 2: No labeling of real measure in Figure. Is it a percentage? What is the difference between bar one and two? One knowledge and second no GDM complication.\n\nFig. 3: No proper labeling. Again is GDM test not a required part of knowledge?\n\nTable 2: Title has to be modified to show that data is in percentage. Why distribution? Language edition required for the indicators. Rather than Ask, Speak etc., can mention availability of BP records, height records, family history records. Leaving ‘–‘ in columns is also confusing. Does it mean 0%, if so show it as 0%.\n\nTable 3: Title needs correction. The mention of 'No' for Number is to be avoided. There is a mix up of % and numbers in the table. For many slots only '_' is shown. Is it 0%?\n\nTable 3 shows that many obstetric complications are not observed in many centers. What is the relevance of this information for GDM? What the authors mean from absence of these complications is not clear. Are no records available? Otherwise it may indicate less severe cases being managed or good obstetric care. The Sample number is not adequate to assess these queries.\n\nTable 4 title is also to be modified. Terms like ‘Insulin Injections’ are inappropriate for a scientific paper. Do you mean Insulin vials?\n\nThe discussion section should have a limitation section highlighting the inadequacies of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "150780",
"date": "04 Oct 2022",
"name": "Micaela Morettini",
"expertise": [
"Reviewer Expertise Metabolism",
"bioengineering"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe presented study aims to investigate the perceptions and practices of gestational diabetes mellitus (GDM) detection and management in a selected sub-district of Bangladesh at different healthcare facility levels. Results could be useful to elucidate the required actions to be planned for the improvement of GDM screening and treatment/follow-up. Overall, the manuscript is well structured but lacks attention to detail in the presentation of the results and in the discussion of the main achievements and it is also cumbersome in some parts of the text. The study sample is quite limited (and this should be at least acknowledged as a limitation of the study) and this does not allow generalization of the results.\n\nThe connection between the last sentence of the first paragraph in the introduction and the rest of the paragraph seems to be missing. I suggest rephrasing.\n\nPlease add relevant references for the statement in lines 8 to 11 of the third paragraph of the introduction.\n\nThe introduction lacks some key information regarding the reference guidelines/practices on GDM detection and management to compare with and determine how good/bad the situation in Bangladesh is managed.\n\nPlease replace the acronym in the titles with a full description.\n\nPlease specify in the data analysis section how quantitative data were cleaned.\n\nIn Figure 2 and Figure 3 measurement units are missing. The layout of Figure 3 should be revised, since the data and bar overlap.\n\nTable 3 legend should be modified in “Distribution of indicators based on record keeping status among the facilities”.\n\nTypo in Table 4 line 3.\n\nThe authors should discuss what the study adds with respect to previous findings.\n\nPlease revise the discussion in order to emphasize the main results with the addition of study limitations.\n\nThe extended data IDI Discussion Guide needs to be translated into English.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-189
|
https://f1000research.com/articles/8-1482/v1
|
22 Aug 19
|
{
"type": "Study Protocol",
"title": "Stage 1 Registered Report: How subtle linguistic cues prevent unethical behaviors",
"authors": [
"Wen Guo",
"Huanxu Liu",
"Jingwen Yang",
"Yuqi Mo",
"Can Zhong",
"Yuki Yamada",
"Wen Guo",
"Huanxu Liu",
"Jingwen Yang",
"Yuqi Mo",
"Can Zhong"
],
"abstract": "Different ways of description can easily influence people’s evaluation and behaviors. A previous study suggested that subtle linguistic differences in ethical reminder instructions can cause differences in preventing readers’ unethical behavior. The present study aims to replicate the previous finding by Bryan and his colleagues (2013) in the Japanese context, additionally exploring the influence of unfamiliar instructions that capture participants’ attention. In two experiments, which are planned to be conducted online, participants are asked to make 10 coin-tosses and report the number of “heads,” indicating the amount of money that could be earned. We will manipulate instructions (“Don’t cheat” vs. “Don’t be a cheater” vs. no instruction as a control) for each participant group including nearly 270 participants (Experiment 1). Next, we will conduct an extended experiment with an additional task in which more attention is directed toward the text (Experiment 2). Through these registered experiments, we examine the credibility of the previous finding that type of instruction affects the occurrence of unethical behaviors.",
"keywords": [
"cheating",
"persuasion",
"attention",
"moral",
"self construal",
"labeling"
],
"content": "Introduction\n\nWhen people behave dishonestly, they usually downplay the seriousness of the dishonest act (e.g., Monin & Jordan, 2009; Steele, 1988), weakening the link between the dishonesty and one’s self-identity (e.g., Bandura, 1999) to avoid the correspondent inference (Jones & Nisbett, 1972; Ross, 1977) that he or she is the kind of person who behaves dishonestly. According to self-concept maintenance theory, individuals strive to create and maintain an image of themselves as good and ethical people (Markus & Wurf, 1987; Mazar et al., 2008).\n\nMeanwhile, different ways of description can easily influence people’s evaluation and judgment about something, even though they have a wealth of previously established knowledge (Fausey & Boroditsky, 2010). For instance, using a transitive verb (agentive description, e.g., “Timberlake ripped the costume”) to describe an accident makes participants significantly more likely to blame the actor, compared to the same description with the words changed to the intransitive verb (nonagentive description, e.g., “The costume ripped”). Another study found that, for children aged 5–7 years old, when a noun label was employed to describe a character (e.g., “She is a carrot-eater”) rather than a verbal predicate (e.g., “She eats carrots whenever she can”), their judgment about those characteristics would be more stable over time (Gelman & Heyman, 1999). The same phenomenon is demonstrated regarding self-perception (Walton & Banaji, 2004). It is possible that language has some effect in this category (Gelman et al., 2000) because when nouns are used to refer to something, one may have a deeper understanding of it, which is noted to “enable inductive inferences” (Gelman & O’Reilly, 1988).\n\nWhen a word description is linked with unethical behaviors and used to categorize who someone is, people act more conservatively. Bryan et al. (2013) study showed that changing the term from “Don’t cheat” to “Don’t be a cheater” can decrease unethical behavior. They manipulated an instruction (e.g. “Please don’t be a cheater” or “Please don’t cheat”) to inhibit participants from conducting unethical behaviors. As a result, the “Don’t be a cheater” group had a significant decrease in unethical cheating behaviors. In another experiment, Bryan et al. (2011) found that more people would choose to vote if they heard the words “be a voter” rather than “to vote” on the day before election day. Additionally, research showed that, compared to “helping,” “being a helper” encouraged more children to conduct kind behaviors toward others (Bryan et al., 2014). However, subsequent research found that although “being a helper” can lead to more kind behaviors initially, once there is a setback, the backfire may also be stronger accordingly (Foster-Hanson et al., 2018). The reason underlying this phenomenon is as follows: as category labels, nouns bear a strong link to identity and may lead to self-doubt once he/she fails. Regarding ethical behavior, a moral-character model has been proposed, where moral character consists of motivation, ability, and identity elements (Cohen & Morse, 2014). Moral identity refers to being disposed toward valuing morality and wanting to view oneself as a moral person. This disposition should be considered when attempting to understand why people who behave unethically tend to apply all kinds of strategies to weaken the behavior-identity link (Bandura, 1999), including “euphemistic labeling” regarding language.\n\nAccording to Bryan et al. (2011), the effect of noun expression is a motivation-driven process. When the noun is involved with positive identities, such as “voter” and “helper,” people produce more correlated behaviors; when the noun is involved with negative identities such as “cheater,” however, people produce fewer correlated behaviors.\n\nIn general, we believe that highlighting a self-identity word will decrease unethical behaviors; for example, according to Blasi (1984), a moral person is one for whom moral categories and moral notions are central, essential, and important to self-understanding. Morals cut deeply to the core of what and who they are as people. However, one study revealed that highly constructed self-identities are associated with more unethical behaviors (Cojuharenco et al., 2012). There are therefore still many unsolved problems about the relationship between the concept of self-identity and unethical behaviors.\n\nIn this study, we aim to replicate Experiment 3 of Bryan et al. (2013), for the following reasons:\n\nFirst, the participants of Experiment 1 in Bryan et al. (2013) were asked to think of a number from 1 to 10. If the number was even, they would be paid $5; if it was odd, there was no reward. The authors found that “participants who were instructed to generate a random number typically show a strong bias toward odd numbers,” yet an accurate number could not be verified at the end of experiment. Thus, we abandoned the method of Experiment 1 because it contains much uncertainty. Compared with Experiment 2, which just constituted by two condition: “cheater” and “cheating”, a baseline group was included in Experiment 3 which made Experiment 3 more complete in its design. Moreover, we found that the effect size of Experiment 3 was small (f = 0.302). With an effect size equaling 0.302 in G*Power (significance level α = 0.05, power level 1-β = 0.95), Experiment 3 required at least 174 participants; in fact, only 99 people joined the original research. From this, we suppose that the effect size in Experiment 3 was overvalued.\n\nAccording to the above review, high levels of self-identity and the willingness of individuals to maintain a positive self-view should decrease unethical behaviors. We anticipate that the results will remain the same as for the original article, in which utilizing the self-relevant noun “cheater” curbed cheating behaviors significantly more than the verb “cheating.”\n\n\nMethods\n\nOur experiment will be conducted online in a private and impersonal way, which means that participants will not meet or be expected to meet the experimenters. We aim to replicate Experiment 3 of Bryan et al. (2013), in which there are three conditions: “cheater,” “cheating,” and “baseline.” In the baseline condition, a reminder about cheating will not be mentioned.\n\nParticipants. Participants will be users of the Yahoo! Crowdsourcing Service in Japan. Participants are required to meet the a priori criterion that they are native Japanese speakers. We plan to conduct a pilot test to determine the shortest time in which one could reasonably participate in the experiment in good faith. This pilot test is detailed in a later section (Outlier extraction). Participants will be excluded if they complete the experiment faster than the pilot test time. Repeat participation will be prevented.\n\nProcedure. Online instructions will explain that a recent controversial article has claimed to report the first scientific evidence for paranormal phenomena.\n\nWe ask the participants to find a coin at home and, while trying to influence the outcome of each toss with their minds, flip the coin 10 times, making it land on “heads” as often as possible. They will be asked to ensure that they are “properly motivated.” For every toss landing on “heads,” they will receive 10 T-points (≒ 10 Yen) as a reward. Noting that the average Yahoo! Crowdsourcing user receives two T-points for one task, the reward we set is five times that amount. Even those participants who report a chance level of five “heads,” they will receive 25 times more points than for a normal task. This reward should be large enough for some participants to conduct a cheating behavior.\n\nParticipants will be randomly assigned into the three conditions. Three conditions are posted on the crowdsourcing website, but a person who participated in one of the conditions cannot participate in the other conditions. This results in passive randomization. In the “cheater” and “cheating” conditions, the manipulation is embedded in the instructions. We referred to the instructions in the original experiment (Bryan et al., 2013) and translated it into Japanese, as follows:\n\nNOTE: Please don’t [cheat/be a cheater] and report that one or more of your coin flips landed heads when it really landed tails! Even a small [amount of cheating/number of cheaters] would undermine the study, making it appear that psychokinesis is real.\n\n[嘘つきにならない/嘘をつかない]でください。コイントスの結果，実際にはコインの裏が出たのに表が出たと報告しないでください。\n\nたとえわずかでも[嘘があると/嘘つきがいると]，この研究を台無しにし，念力が実在するように見せてしまいます。\n\nThe manipulation is also embedded in the instructions on the next page, where participants log the outcomes of their 10 coin-flips. At the top of the page, a message is displayed in large red words: “嘘つきにならないでください/嘘をつかないでください” Which was “PLEASE DON’T [CHEAT/ BE A CHEATER]” as in the original experiment (Bryan et al., 2013).\n\nIn the baseline condition, the instructions are the same as above, except that the cheating message is not mentioned.\n\nPower analysis and sample size. Because Experiment 3 of Bryan et al. (2013) did not report the effect size of η2, first, we calculated the effect size of the analysis of variance (ANOVA) result from the F and df values. Bryan et al., 2013 reported the statistics of their one-way ANOVA as F(2, 96) = 4.38, p = .015. Hence, we calculated η2, based on Cohen’s (1973) method, as η２＝.0836. Then, we calculated the effect size f as follows: f = √(η2/(1 – η2) = 0.302. The small sample size may overestimate the effect size so, as a replication convention (e.g., Nitta et al., 2018), we halved the effect size of the original experiment, and used G*Power 3.1.9.3 (Faul et al., 2009) to conduct a power analysis (i.e., to 0.151). In G*Power, we set the significance level α = 0.05, power level 1-β = 0.95, and effect size f = 0.151. According to the conditions of the original experiment, we will divide the participants into three groups. The required total sample size is 681, with 227 participants in each group. Therefore, we will try to recruit at least 681 participants. Data collection will not exceed 810 participants. This stopping rule is set because it is difficult for us to control the number of participants to just 681, due to the characteristics of the simultaneous participatory online recruitment system; therefore, we will allow for up to 120% of the required sample size (i.e., 810). If more than 810 people participate in the experiment, we will select the data of the first 810 participants based on the time stamp and use this for the analysis. Also, we set the number of participants (max. 365 males and 445 females) to match the gender distribution of the original study (male: female = .45: .55).\n\nData analyses. In this study, the dependent variable is the mean number of “heads” reported. In the original experiment, a one-way ANOVA and t-test will be performed. Specifically, the ANOVA will be performed for analyzing the main effect of the three groups. A problem in the original study was that it did not adjust for any significance level in subsequent multiple comparisons. Therefore, in the present study, we will use a one-way ANOVA and Tukey’s method for the multiple comparisons. Additionally, in order to check the cheating in each group, the original study performed one-sample t-tests between the mean number of “heads” reported and the chance level (i.e., 50%). These analyses will be performed using jamovi (version 1.0.5). The original results are summarized in Table 1.\n\nOutlier extraction. For our online experiment, we will establish a minimum completion time (MCT) for inclusion in the final sample. By asking five colleagues who are unfamiliar with this experiment to complete the experiments as fast as possible, then calculating the mean completion time. Specifically, each colleague will perform a coin toss ten times. After each toss they record on the website that will be used in the experiment. This pilot test will not include the work that “influence the outcome of each toss with their ‘properly motivated’” and measures only the required time of the coin toss and recording. Bryan et al. (2013) also used the MCT as an extraction criterion. We will exclude those participants who complete it faster than this MCT, because they may rush through the experiment and fail to complete it in good faith.\n\nThis experiment will be employed as an extended, conceptual replication of Experiment 3 in the original study (Bryan et al., 2013). If our Experiment 1 successfully replicates the previous experiment, we will conduct Experiment 2, adding a “cheating” condition in which we use tasks concerning an instruction to ensure that participants’ attention is captured (e.g., Folk et al., 1992; Folk et al., 2002). There are two main reasons why we wish to conduct Experiment 2. First, some studies have revealed that a moral reminder may be an effective way to decrease unethical behaviors (Belle & Cantarelli, 2017). While there was no difference in the rate of cheating between the baseline and the “Don’t cheat” groups in Experiment 3 of Bryan et al. (2013), their results suggest that the ethical reminder in the “Don’t cheat” condition did not work. Because those experiments were conducted online, it is difficult to ensure whether the participants had actually seen and understood the instruction. Otherwise, it is also possible that the participants ignored the original experiment’s instruction due to satisficing (e.g., Chandler et al., 2014; Oppenheimer et al., 2009; Sasaki & Yamada, 2019). This may be the reason that there was no significant difference between the cheating and baseline conditions. The second reason to conduct Experiment 2 is that the significance of the cheater condition may occur because of excessive attention being paid to the instruction. Noticeably, the main difference between our Experiment 1 and the original Experiment 3 lies in the different language used in the instruction. Thus, if our Experiment 1 is a successful replication, we will then choose to focus on the expression of the Japanese instruction, rather than the English instruction of the original Experiment 3.\n\nTo confirm this assumption, we conducted a preliminary experiment, asking participants to evaluate their familiarity with certain expressions in Japanese. The expressions “Don’t cheat” and “Don’t be a cheater” were translated into Japanese and native speakers evaluated their familiarity (1: not familiar to 5: very familiar) with them via an Internet survey on the Yahoo! Crowdsourcing Service. The protocol of this experiment was registered on the Open Science Framework (Guo et al., 2019). The results showed that the familiarity rating score in the “cheater” condition was significantly lower than that in the “cheating” condition, t(64) = 6.73, p < .001, Cohen’s d = 0.834. Hence, we conjecture that the anticipated difference in the results between the “cheating” and “cheater” conditions in Experiment 1 may partly occur due to differences in attention paid to the instruction, instead of the preservation of a positive self-image proposed by the previous study (Bryan et al., 2013). This means that part of the effect of the “cheater” condition is due to the unfamiliar expression, which attracts people’s attention then plays a role in preventing them from conducting unethical behavior. See Extended data for details about this experiment.\n\nIn our Experiment 2, we manipulated the way in which participants saw the instructions to explore the differences between the cheating and baseline conditions. Experiment 2 comprises three conditions: “cheating,” “cheating” with task, and baseline. We predict that the cheating with task condition will be more effective in curbing unethical behaviors than the baseline condition, because the task will arouse more attention. While the instruction in the cheating condition will be in large red capital letters, this should have no significant difference compared with baseline.\n\nProcedure. The procedure for Experiment 2 is identical to that of Experiment 1, except for important differences in two aspects. In Experiment 2, we will focus on whether the participants read the instructions as diligently as we expect. First, we will delete the original “cheater” condition and add another “cheating” condition. Second, in the new “cheating” condition, we will add a task page in which participants are asked to choose the exact expression (i.e., “Don’t cheat”) that appeared on the screen from three sample sentences. We will remind participants of this task in advance to ensure they read the instructions carefully.\n\nPower analysis and participants. Because the power analysis of Experiment 2 is the same as in Experiment 1, we intend to recruit participants in the same way as Experiment 1. The minimum completion time will also be established for participants to be included in the final sample. The exclusion standard is similar to Experiment 1.\n\nData analyses. In Experiment 2, the dependent variable is the mean number of “heads” reported. We will still use a one-way ANOVA and Tukey’s method for the multiple comparisons. To check the cheating rate in each group, a one-sample t-test between the mean number of “heads” reported and the chance level (50%) will be analyzed.\n\n\nStudy timeline\n\nCurrently, online experiments for participants to conduct the coin-toss task are under construction. After stage 1 acceptance, our colleagues will be asked to complete the pilot test to calculate the MCT. Then, we will post our experiments on the Yahoo! Crowdsourcing Service to recruit participants. We are supposed to complete the experiments and subsequent analysis within two months.\n\nThe present study received approval from the psychological research ethics committee of the Faculty of Human-Environment Studies at Kyushu University (approval number: 2019-004). Completion of experiments will be taken as consent to participate from participants. Participants have the right to withdraw from the experiment at any time without providing a reason. In addition, we will protect participants’ personal information. Because this study will be conducted online, even if participants engage in cheating behaviors, we cannot identify them or meet the participants face-to-face.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework: How subtle linguistic cues prevent unethical behaviors, https://doi.org/10.17605/OSF.IO/68FVK (Guo et al, 2019).\n\nThis project contains the following extended data:\n\n- Protocol for the pilot study conducted for Experiment 2.\n\n- Data collected for the pilot study conducted for Experiment 2.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThis research was supported by JSPS KAKENHI (15H05709, 16H01866, 17H00875, 18H04199, and 18K12015).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBandura A: Moral disengagement in the perpetration of inhumanities. Pers Soc Psychol Rev. 1999; 3(3): 193–209. PubMed Abstract | Publisher Full Text\n\nBelle N, Cantarelli P: What Causes Unethical Behavior? A Meta-Analysis to Set an Agenda for Public Administration Research. Public Adm Rev. 2017; 77(3): 327–339. Publisher Full Text\n\nBlasi A: Moral identity: Its role in moral functioning. Morality, Moral Behavior, and Moral Development. 1984; 128–139.\n\nBryan CJ, Adams GS, Monin B: When cheating would make you a cheater: implicating the self prevents unethical behavior. J Exp Psychol Gen. 2013; 142(4): 1001–5. PubMed Abstract | Publisher Full Text\n\nBryan CJ, Master A, Walton GM: \"Helping\" versus \"being a helper\": invoking the self to increase helping in young children. Child Dev. 2014; 85(5): 1836–1842. PubMed Abstract | Publisher Full Text\n\nBryan CJ, Walton GM, Rogers T, et al.: Motivating voter turnout by invoking the self. Proc Natl Acad Sci U S A. 2011; 108(31): 12653–12656. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChandler J, Mueller P, Paolacci G: Nonnaïveté among Amazon Mechanical Turk workers: consequences and solutions for behavioral researchers. Behav Res Methods. 2014; 46(1): 112–130. PubMed Abstract | Publisher Full Text\n\nCohen J: Eta-squared and partial eta-squared in fixed factor ANOVA designs. Educ Psychol Meas. 1973; 33(1): 107–112. Publisher Full Text\n\nCohen TR, Morse L: Moral character: What it is and what it does. Res Organ Behav. 2014; 34: 43–61. Publisher Full Text\n\nCojuharenco I, Shteynberg G, Gelfand M, et al.: Self-construal and unethical behavior. J Bus Ethics. 2012; 109(4): 447–461. Publisher Full Text\n\nFaul F, Erdfelder E, Buchner A, et al.: Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods. 2009; 41(4): 1149–1160. PubMed Abstract | Publisher Full Text\n\nFausey CM, Boroditsky L: Subtle linguistic cues influence perceived blame and financial liability. Psychon Bull Rev. 2010; 17(5): 644–650. PubMed Abstract | Publisher Full Text\n\nFolk CL, Leber AB, Egeth HE: Made you blink! Contingent attentional capture produces a spatial blink. Percept Psychophys. 2002; 64(5): 741–753. PubMed Abstract | Publisher Full Text\n\nFolk CL, Remington RW, Johnston JC: Involuntary covert orienting is contingent on attentional control settings. J Exp Psychol Hum Percept Perform. 1992; 18(4): 1030–44. PubMed Abstract | Publisher Full Text\n\nFoster-Hanson E, Cimpian A, Leshin RA, et al.: Asking Children to \"Be Helpers\" Can Backfire After Setbacks. Child Dev. 2018; 1–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGelman SA, O'Reilly AW: Children's inductive inferences within superordinate categories: the role of language and category structure. Child Dev. 1988; 59(4): 876–887. PubMed Abstract\n\nGelman SA, Heyman GD: Carrot-eaters and creature-believers: The effects of lexicalization on children's inferences about social categories. Psychol Sci. 1999; 10(6): 489–493. Publisher Full Text\n\nGelman SA, Hollander M, Star J, et al.: The role of language in the construction of kinds. Psychol Learn Motiv. 2000; 39: 201–263. Publisher Full Text\n\nGuo W, Liu H, Yang J, et al.: How subtle linguistic cues prevent unethical behaviors. 2019. http://www.doi.org/10.17605/OSF.IO/68FVK\n\nJones E, Nisbett R: The actor and the observer: Divergent perceptions of the causes of behavior. Attribution: Perceiving the Causes of Behavior. 1972; 79–94.\n\nMarkus H, Wurf E: The dynamic self-concept: A social psychological perspective. Annu Rev Psychol. 1987; 38(1): 299–337. Publisher Full Text\n\nMazar N, Amir O, Ariely D: The dishonesty of honest people: A theory of self-concept maintenance. J Mark Res. 2008; 45(6): 633–644. Publisher Full Text\n\nMonin B, Jordan A: The dynamic moral self: A social psychological perspective. Personality, Identity, and Character: Explorations in Moral Psychology. 2009; 341–354. Publisher Full Text\n\nNitta H, Tomita H, Zhang Y, et al.: Disgust and the rubber hand illusion: a registered replication report of Jalal, Krishnakumar, and Ramachandran (2015). Cogn Res Princ Implic. 2018; 3(1): 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOppenheimer DM, Meyvis T, Davidenko N: Instructional manipulation checks: Detecting satisficing to increase statistical power. J Exp Soc Psychol. 2009; 45(4): 867–872. Publisher Full Text\n\nRoss L: The intuitive psychologist and his shortcomings: Distortions in the attribution process. In Adv Exp Soc Psychol. 1977; 10: 173–220. Publisher Full Text\n\nSasaki K, Yamada Y: Is crowdsourcing suitable for investigating visual perception? A case of contrast sensitivity. Manuscript Submitted for Publication. 2019.\n\nSteele CM: The psychology of self-affirmation: Sustaining the integrity of the self. In Adv Exp Soc Psychol. 1988; 21: 261–302. Publisher Full Text\n\nWalton GM, Banaji MR: Being what you say: The effect of essentialist linguistic labels on preferences. Soc Cogn. 2004; 22(2): 193–213. Publisher Full Text"
}
|
[
{
"id": "55680",
"date": "24 Oct 2019",
"name": "Michiko Asano",
"expertise": [
"Reviewer Expertise Cognitive 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\nSummary\n\nThis study is designed to examine an interesting topic of whether and how subtle linguistic cues may prevent unethical behaviors. The proposal is generally well written. However, I have some concerns, which I address below.\nIntroduction: It is not clear to me why the authors decided to replicate Bryan et al. (2013)1 among many other studies. I understand from the proposal the reason why they chose Experiment 3 of Bryan et al. (2013) over Experiment 1 or 2. I also understand that the sample size in Experiment 3 of Bryan et al. was not sufficient and thus the results may require replication with a larger sample. However, I do not understand the importance of replicating their results in addressing the research question of whether and how subtle linguistic cues prevent unethical behaviors. The authors should elaborate on this issue to clarify the significance of their study (e.g., “It is important to replicate Bryan et al.’s study as it has a significant impact on this field.” or “The protocol of Experiment 3 is useful and applicable to future studies. Therefore, the robustness of their findings should be tested carefully).\n\nPlease state in the second to last paragraph of the Introduction that, unlike Experiment 1, Bryan et al. (2013) used a coin-tossing task in Experiments 2 and 3. Otherwise, this paragraph is hard to follow.\n\nThe procedure of Experiment 1 in the current study is not described sufficiently. Please state that the cover story (examination of a paranormal phenomenon) is exactly the same as that of Experiment 3 of Bryan et al. (2013).\nIt is also necessary to specify how the authors will explain to their participants about the relationship between the coin flips and the amount of reward. Bryan et al. (2013) used the following description: \"The instructions acknowledged that the laws of probability dictate that people would, on average, make $5, although some would ‘make as much as $10 just by chance’ and others would ‘make as little as $0’”. If the procedure is identical to that of Experiment 3 of Bryan et al. (2013) except for the use of the Japanese language, stating so would help readers understand the protocol.\n\nThe authors list the following two points as the reason why they plan to direct participants’ attention toward the ethical reminder in Experiment 2. (a) First, there was no significant difference in the rate of cheating between “don’t cheat” and the baseline (i.e., no ethical reminder) conditions in the study by Bryan et al. The authors conjecture that participants in Bryan et al.’s did not pay sufficient attention to the ethical reminder. (b) Second, Their preliminary experiment has revealed that the Japanese translation of the expression “don’t be a cheater” (嘘つきにならないで), compared to that of “don’t cheat” (嘘をつかないで), is less familiar to Japanese speakers and thus may attract more attention. Such difference in familiarity may produce results similar to Experiment 3 of Bryan et al. (2013), irrespective of whether the expression bears a strong link to self-identity or to the action. To address these concerns, the authors are planning to add “cheating (’don’t cheat’) with task” condition, in which they test whether participants paid attention to the ethical reminder.\n\nAs for (a), I am not sure whether the lack of difference in cheating rates between “don’t cheat” and the baseline conditions in Bryan et al.’s study necessarily means that the participants did not pay attention to the ethical reminder. The cheating rate was significantly lower in “don’t be a cheater” condition than in the “don’t cheat” condition in their study. Doesn’t this mean that the participants paid sufficient attention to the ethical reminder?\nAs for (b), I agree with the authors that ethical reminders that attract more attention may prevent unethical behaviors more strongly. However, I do not understand what hypothesis the authors will test using the protocol of Experiment 2. Are they trying to show that it is attention to ethical reminders, rather than the linguistic expression linked to self-identity, that prevents unethical behaviors? If so, how do they interpret the results of Bryan et al. (2013)?\n\nI do not understand the task of the “cheating with task” condition in Experiment 2. Will the authors use this task to classify participants into those who paid attention to the ethical reminder and those who did not, and analyze the cheating rate separately for these two groups? Or is the task just for bringing participants’ attention to the ethical reminder before the coin-tossing task?\n\nHave the authors pre-specified sufficient outcome-neutral tests for ensuring that the results obtained can test the stated hypotheses, including positive controls and quality checks? Partly\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No",
"responses": [
{
"c_id": "5113",
"date": "24 Dec 2019",
"name": "Yuki Yamada",
"role": "Author Response",
"response": "Thank you very much for taking the time and effort to check our manuscript. we also appreciate your helpful comments. Individual answers are given below. 1. Introduction: It is not clear to me why the authors decided to replicate Bryan et al. (2013)1 among many other studies. I understand from the proposal the reason why they chose Experiment 3 of Bryan et al. (2013) over Experiment 1 or 2. I also understand that the sample size in Experiment 3 of Bryan et al. was not sufficient and thus the results may require replication with a larger sample. However, I do not understand the importance of replicating their results in addressing the research question of whether and how subtle linguistic cues prevent unethical behaviors. The authors should elaborate on this issue to clarify the significance of their study (e.g., “It is important to replicate Bryan et al.’s study as it has a significant impact on this field.” or “The protocol of Experiment 3 is useful and applicable to future studies. Therefore, the robustness of their findings should be tested carefully). Reply: In the present replication study, we are planning to test whether instruction has any effect on the outcome of the experiments. Psychological researchers use instruction before an experiment to explain the aims of the experiment and what participants should do in the experiment. However, up to now it has remained unclear whether these instructions affect the performance of participants. In psychological experiments, one of the problems that affects the results is expectancy effects or participants’ laziness. Laziness is a particularly frequent phenomenon, seeing participants try to finish the experiment as easily as possible just to obtain rewards. Sometimes there is even cheating. There is much evidence suggesting a relationship between reminders and behaviors in the field of social psychology (e.g., Johns, Schmader, & Martens, 2005; Ling, Beenen et al., 2005; Bryan, Master, & Walton, 2014; Bryan, Walton, Rogers, & Dweck, 2011). In particular, Bryan et al. (2013) reported for the first time that a subtle difference in instruction affects cheating. For the reasons above, the results of Bryan et al. are really important for conducting psychological research. Therefore, it is necessary to confirm the reliability of their finding. In fact, another lab has already tried to replicate Bryan et al., although they were not pre-registered (Savir, 2019). In Experiment 1 of Bryan et al., participants were asked to think of a number from 1 to 10; if the number was even, they would be paid $5, and otherwise, they would gain no reward. We abandoned this method because it leads to much uncertainty. First, Bryan et al. cited previous research results indicating that participants instructed to generate a random number typically show a strong bias toward odd numbers (Kubovy & Psotka, 1976), so they intentionally paid for even numbers. However, this oddness bias has not been confirmed for betting behaviors. Secondly, thinking of a number is an imaginary event occurring in one’s inside world, not a real external event. Hence, it is suboptimal as an index of falsification, as the index used for cheating should emphasize that the cheating participants’ reports differ from the fact. Therefore, in our experiments, we use the coin-tossing task in Experiments 2 and 3 of Bryan et al. as a method of measuring cheating behavior (falsifying the coin-toss result). Furthermore, compared with Experiment 2 of Bryan et al. which just consisted of two conditions, a baseline group was included in Experiment 3, which made it more complete in design. Therefore, we decided to replicate Experiment 3 of Bryan et al. in our study. We amended the introduction to make these points clearer. Thank you for your suggestions, which have helped us clarify the purpose of the present replication. 2. Please state in the second to last paragraph of the Introduction that, unlike Experiment 1, Bryan et al. (2013) used a coin-tossing task in Experiments 2 and 3. Otherwise, this paragraph is hard to follow. Reply: As a response to this suggestion, we added the following sentences: “In [Bryan et al.’s] Experiments 2 and 3, they used a coin-tossing task: participants were asked to toss a coin and receive a reward corresponding to the result of their coin flips. We choose this method for our experiment because tossing a coin induces a real external event, which is more objective and operable, and hence it is better than thinking of a number to measure cheating behavior.” 3.The procedure of Experiment 1 in the current study is not described sufficiently. Please state that the cover story (examination of a paranormal phenomenon) is exactly the same as that of Experiment 3 of Bryan et al. (2013). It is also necessary to specify how the authors will explain to their participants about the relationship between the coin flips and the amount of reward. Bryan et al. (2013) used the following description: \"The instructions acknowledged that the laws of probability dictate that people would, on average, make $5, although some would ‘make as much as $10 just by chance’ and others would ‘make as little as $0’”. If the procedure is identical to that of Experiment 3 of Bryan et al. (2013) except for the use of the Japanese language, stating so would help readers understand the protocol. Reply: In the original experiment, Bryan et al. referred to the article by Bem (2011, which had received considerable media attention) to describe a recent discovery of evidence for paranormal phenomena. We will use the same article for our instruction. As the details of the paranormal article are not given in Bryan et al.’s paper, we will abstract the content of Bem’s article and translate it into Japanese and add this part to our instructions. As for the relationship between the coin flips and reward, we will instruct participants that they will receive a reward corresponding to the result of their coin flips. In the Yahoo! Crowdsourcing Service, we gave a reward of about 5 times the average, to encourage cheating. However, the rewards that each person ultimately gets still depend on the result of their coin flips. 4, The authors list the following two points as the reason why they plan to direct participants’ attention toward the ethical reminder in Experiment 2. (a) First, there was no significant difference in the rate of cheating between “don’t cheat” and the baseline (i.e., no ethical reminder) conditions in the study by Bryan et al. The authors conjecture that participants in Bryan et al.’s did not pay sufficient attention to the ethical reminder. (b) Second, Their preliminary experiment has revealed that the Japanese translation of the expression “don’t be a cheater” (嘘つきにならないで), compared to that of “don’t cheat” (嘘をつかないで), is less familiar to Japanese speakers and thus may attract more attention. Such difference in familiarity may produce results similar to Experiment 3 of Bryan et al. (2013), irrespective of whether the expression bears a strong link to self-identity or to the action. To address these concerns, the authors are planning to add “cheating (’don’t cheat’) with task” condition, in which they test whether participants paid attention to the ethical reminder. As for (a), I am not sure whether the lack of difference in cheating rates between “don’t cheat” and the baseline conditions in Bryan et al.’s study necessarily means that the participants did not pay attention to the ethical reminder. The cheating rate was significantly lower in “don’t be a cheater” condition than in the “don’t cheat” condition in their study. Doesn’t this mean that the participants paid sufficient attention to the ethical reminder? As for (b), I agree with the authors that ethical reminders that attract more attention may prevent unethical behaviors more strongly. However, I do not understand what hypothesis the authors will test using the protocol of Experiment 2. Are they trying to show that it is attention to ethical reminders, rather than the linguistic expression linked to self-identity, that prevents unethical behaviors? If so, how do they interpret the results of Bryan et al. (2013)? Reply: Thank you for your comments. We will answer your questions one by one. As for (a), I am not sure whether the lack of difference in cheating rates between “don’t cheat” and the baseline conditions in Bryan et al.’s study necessarily means that the participants did not pay attention to the ethical reminder. The cheating rate was significantly lower in “don’t be a cheater” condition than in the “don’t cheat” condition in their study. Doesn’t this mean that the participants paid sufficient attention to the ethical reminder? Possibly our description in the draft was not clear enough. In fact, we did not discuss participants’ attention in the original experiment. However, in our replication experiment, we realized that the attention of the participants to the reminder may become a problem, and aimed to explore this. As your comment reflects, the existing evidence is not sufficient to address this problem, nor, of course, can our experiments clarify the problem of attention among the participants in the original experiment. Based on result of our Experiment 2, participants’ attention to reminders in the original experiment will be discussed in the General discussion section of the paper (which is not included in the Stage 1 manuscript). Hence, we removed reason (a) in our draft and instead described the reason why our Experiment 2 had to highlight the issue of attention. We made the following changes to the manuscript: “When we translated the instruction into Japanese, we felt the unfamiliarity of a “cheater” condition in a Japanese language situation. Participants in our experiment may find that the reminder “not to be a cheater” captures extra attention because of this sense of deviation. Therefore, even if the result of the original experiment is completely reproduced in our Experiment 1, it will not fully support the finding of the original experiment, as the reason for the possible different dishonest behavior rates between the “cheating” and “cheater” conditions in our Experiment 1 may be that the participants in the “cheating” group paid relatively less attention to the instruction, so that “cheating” weakly worked as a moral reminder in this condition. Because the experiments are conducted online, it is difficult to ensure that the participants have actually seen and understood the instruction; in addition, it is also possible that the participants ignored the instruction of our Experiment 1 due to satisficing, (e.g., Chandler et al., 2014; Oppenheimer et al., 2009; Sasaki & Yamada, 2019), further diminishing the effect of the unattended reminder (i.e., “cheating”).” As for (b), I agree with the authors that ethical reminders that attract more attention may prevent unethical behaviors more strongly. However, I do not understand what hypothesis the authors will test using the protocol of Experiment 2. Are they trying to show that it is attention to ethical reminders, rather than the linguistic expression linked to self-identity, that prevents unethical behaviors? If so, how do they interpret the results of Bryan et al. (2013)? We will mainly discuss reason (b). We hypothesized that there is a difference in the dishonest behavior rate between the “cheating” with task condition and baseline in Experiment 2 based on the premise of the results of the preliminary experiment (i.e., the familiar expressions used in the two reminders are certainly different). In order to convey the relationship between the preliminary experiment and the hypothesis more clearly, we made the following amendments to the manuscript: “Our Experiment 2 will only be performed when the results of Experiment 1 successfully replicate those of the original experiment. We will conduct Experiment 2, adding a “cheating” condition in which we use tasks concerning an instruction to ensure that participants’ attention is captured.” In this way, we want to find out if our experiments really support (or do not support) the results of the original experiment. 5, I do not understand the task of the “cheating with task” condition in Experiment 2. Will the authors use this task to classify participants into those who paid attention to the ethical reminder and those who did not, and analyze the cheating rate separately for these two groups? Or is the task just for bringing participants’ attention to the ethical reminder before the coin-tossing task? Reply: We intend to use this task only to make sure that participants pay enough attention to the ethical reminder. We will delete the data of participants who fail to give the right answer to the attention task. References Bem, D. J. (2011). Feeling the future: experimental evidence for anomalous retroactive influences on cognition and affect. Journal of personality and social psychology, 100(3), 407. Bryan, C. J., Master, A., & Walton, G. M. (2014). “Helping” versus “being a helper”: Invoking the self to increase helping in young children. Child Development, 85(5), 1836-1842. Bryan, C. J., Walton, G. M., Rogers, T., & Dweck, C. S. (2011). Motivating voter turnout by invoking the self. Proceedings of the National Academy of Sciences, 108(31), 12653-12656. Johns, M., Schmader, T., & Martens, A. (2005). Knowing is half the battle: Teaching stereotype threat as a means of improving women's math performance. Psychological Science, 16(3), 175-179. Ling, K., Beenen, G., Ludford, P., Wang, X., Chang, K., Li, X., ... & Resnick, P. (2005). Using social psychology to motivate contributions to online communities. Journal of Computer‐Mediated Communication, 10(4), 00-00. Savir, T., & Gamliel, E. (2019). To be an honest person or not to be a cheater: Replicating the effect of messages relating to the self on unethical behaviour. International Journal of Psychology, 54(5), 650-658. In this way, your thoughtful and constructive comments have greatly improved our manuscript. We express our sincere gratitude again. We would appreciate it if you approved this manuscript."
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https://f1000research.com/articles/8-1482
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https://f1000research.com/articles/9-181/v1
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11 Mar 20
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{
"type": "Research Article",
"title": "Knowledge, attitude and prevention behavior related to HIV/AIDS among students of a college in Lao-PDR: a cross-sectional study",
"authors": [
"Ranee Wongkongdech",
"Souksathaphone Chanthamath",
"Adisorn Wongkongdech",
"Souksathaphone Chanthamath",
"Adisorn Wongkongdech"
],
"abstract": "Background: Inadequate knowledge, negative attitudes towards people living with HIV/AIDS and risky practices are related to the spread of HIV infection. This study aimed to explore and test associations between knowledge, attitude and AIDS prevention behavior among students in Kham Mouane Technical-Vocation College, Lao People’s Democratic Republic. Methods: A cross-sectional, self-report questionnaire study was conducted in May 2018. Pearson's Chi-squared test and binary logistic regression was used to test the association between variables. Results: Of 939 participants, it was found that: 61.6% were women; the mean age was 20.7 years (range 18-28 years); 40.2% lived in the college dormitory; >80% drank alcoholic beverages; 31.9% drank until slightly intoxicated; 13.7% drank until drunk; and the average drinking rate was 1-2 times a month (50%). Knowledge levels about the route of HIV transmission and how it can be controlled were poor. Misunderstood issues included: HIV transmission can be controlled by having a single partner and using condoms while having sex was correctly understood by only 22.8%; 72% incorrectly reported that HIV can be transmitted through a mosquito bite; and 48.2% believed incorrectly that drinking alcohol can disinfect HIV. Attitudes towards HIV were at a moderate level: the participants believed that infected persons should not reveal themselves in public, at work or college, and that requesting a boyfriend use a condom showed mistrust. HIV prevention practices were reported at a better level: reducing opportunity for sex by not cohabiting or staying privately with a lover and wearing condoms regularly during sexual intercourse, although more than 70% still drank alcohol when visiting nightclubs, associated with an increased likelihood of unprotected sex. Conclusions: Public and community stakeholders need to design HIV prevention classes suited to college environments to improve students’ knowledge, attitudes and risky behavior practices related to HIV.",
"keywords": [
"Knowledge Attitude and Practice",
"Risk behavior",
"HIV",
"College Adolescents",
"Lao PDR"
],
"content": "Introduction\n\nLao People’s Democratic Republic (PDR) is committed to the goal of the World Health Organization’s global strategy for eradicating HIV by 2030 [WHO, 2019], with external budget funds available for addressing HIV in high risk groups. Although the Lao PDR HIV infection prevalence among individuals aged 15 years or older was relatively low at 0.3% (0.2–0.3) in 2017 [UNAIDS, 2018] and high rates of condom use were reported among sex workers (91.8%), there are many potentially serious challenges for HIV prevention action. In 2017, estimates of people living with HIV (PLHIV) over 15 years old increased significantly to 11,000 (9,900–13,000), but anti-retroviral (ART) coverage was estimated at less than half (47%) [HIV and AIDS Data HUB, 2018]. It is estimated that only one-in-four knew their HIV status, leading to delayed treatment and an increased risk of exposing others to HIV infection, long-term complications and deaths due to AIDS [Bowring et al., 2015]. Further, continuing stigma and discrimination has created barriers for at-risk populations to access HIV screening, prevention, treatment and care [Bowring et al., 2015]. It was found from behavior surveillance surveys in 2017 [UNAIDS, 2018] that prevention programs for female sex workers only achieved 50% coverage. In addition, condom use in men who have sex with men was only 25.7%, with only 10.2% of this population aware of their HIV status and only 7.8% accessing care programs [Lao Social Indicator Survey II 2017–18; UNFPA, 2017]. In addition, “kathoys” (biological males who self-identify as female) reported a 27.4% consistent condom use rate, and just 24.1% reported a consistent condom use rate with non-regular partners in the last three months [Phimphachanh & Sayabounthavong, 2004]. Many adolescents reported having sex before 15 years of age, and 6% reported having had anal sex [Sychareun et al., 2013]. Furthermore, Lao PDR is surrounded by four countries (Thailand, Viet Nam, China and Myanmar) that have been reported to be the main contributors to the rising incidence rate of HIV in some provinces of Lao PDR, which together contribute 95% of new infections in the Asia-Pacific region [Silakoune, 2017].\n\nKhammouane Province is located in south-central Lao PDR and is close to the Thai border. In 2016, 271 PLHIV were reported, 92% of whom were in the over 15-year-old group, nearly half were new patients in that year, and 90% had access to ART. The latest figures reported January–August 2017 showed a 14% increase in PLHIV, and new patients increased by 22% [Lao Statistics Bureau, 2018]. The Lao Social Indicator Survey II: 2017 [Lao Statistics Bureau, 2018] reported that for men with multiple partners, 44.7% of 15–24 years (n=253) have had sex before, 61.0% of whom reported condom use during the last sexual intercourse with a non-spouse, non-cohabiting partner in the last 12 months. In total, 27% of 15–19-year-old women had sexual intercourse during the previous 12 months, and 6% had anal sex during the previous 12 months [Phrasisombath et al., 2012], rising to 70% of women who had sex in previous 12 months for the 20–24-year-old group. Furthermore, alcohol consumption can lead to increased chance of unprotected risky sex and it was reported that 90% drank alcohol [Lao Statistics Bureau, 2018]. In addition, studies in high school students have found there was adequate knowledge about HIV, but negative attitudes towards HIV/AIDS and risky, unsafe sex practices [Kosay et al., 2008; Thanavanh et al., 2013].\n\nThis study was undertaken in a Lao PDR tertiary technical-vocational college. To the authors’ knowledge, there has been no prior Lao PDR study of HIV at-risk groups in tertiary colleges. The study aimed to 1) identify student demographics, the level of knowledge of HIV/AIDS, attitudes towards HIV/AIDS and HIV prevention behavior and 2) test associations between the students’ level of knowledge, attitudes and practices regarding HIV/AIDS and its prevention, in order to provide information for agencies to design HIV prevention interventions and evaluate future program operations in this population.\n\n\nMethods\n\nThe study used a cross-sectional self-report questionnaire survey design and was carried out in Kham Mouane Technical-Vocational College, based in Thakhek, the major town in Kham Mouane Province. This urban-based college has a yearly enrolment of around 3,000 students. It offers more than 21 vocational courses, including daytime and evening classes across three academic years.\n\nAn initial sample size of 750 participants was calculated, based on the total student population of 2734, with the confidence level set at 95%, and a variance value of 0.82 (Mayuree & Pannee, 2015). In case of a high non-participation rate due to the sensitivity of the issue, we included approximately 20% more participants, with a final sample size of 939 participants. A list of 2,734 student names was obtained and of these, participants were selected by simple random sampling (939 names were drawn), ensuring that the same proportion of each academic year was invited. All participants invited to the study volunteered to participate.\n\nThe researcher (SC) explained the purpose of the study to all potential participants, their rights to participate and assured them that their information would be kept confidential. Consent forms were handed out and signed by participants or the parents of those aged less than 18 years for participation in the study. Data collection was conducted using the self-report questionnaire and participants completed it individually during an hour break in their classes. All questionnaires were returned to the researchers the next day. Exclusion criterion was failure to give consent. All participants received a small souvenir (a pen) after data collection (participants were not aware they would receive this souvenir prior to participation). All recruitment and data collection took place between 15th–25th May 2018.\n\nThe Knowledge, Attitude and Practice (KAP) survey questionnaire for HIV/AIDS prevention behavior was modified from previous research [Kosay et al., 2008; Thanavanh et al., 2013] and was initially developed in Thai. To assure content validity, it was checked by an expert in health behavior from Khon Kaen University, an expert in AIDS prevention and treatment from the Center for HIV/AIDS/ STIs of Khammoune Province and a teacher from Kham Mouane Technical-Vocational College. It was translated into Lao by the second author (SC), who is fluent in Thai and Lao, and then back-translated from Lao to Thai by another health worker at Khammoune Hospital. An assessment of clarity, feasibility and appropriateness was carried out with 30 additional participants who studied cultivation in a college located 25 km from the study site. We pre-tested and edited three times and finally, it had a Conbrach’s Alpha Reliability Coefficient of 0.75.\n\nThe questionnaires were scored as follows: 1) responses to the knowledge of HIV/AIDS section was recorded through answers of true or false and the level of knowledge of HIV/AIDS was classified as ‘low’ for mean scores of less than 60% correct, ‘medium’ between 60–80%, and ‘high’ over 80% correct (Bloom et al., 1971); 2) responses to the attitudes towards people living with HIV/AIDS section were recorded using a Likert scale of strongly agree (5) to strongly disagree (1) and the level of attitudes towards people living with HIV/AIDS was defined as ‘low’ when the mean score was less than 2.7, ‘medium’ when the mean score was between 2.71–3.7 and ‘high’ when the mean score was over 3.7 (Likert, 1961); 3) responses to the HIV/AIDS prevention practices section were recorded using a Likert scale of 5 (regularly) to 1 (never) and the level of practice was defined as ‘safe’ for mean score over 4.2 and ‘risky’ for those scoring less than 4.2 (Best, 1981). When testing the association between HIV knowledge and HIV attitudes and practices by binary logistic regression, Level of knowledge was defined as ‘low’ for scores up to 50% (0–9.0) and high for those more than 50% (9.1–18.0), level of attitudes was defined as ‘positive’ for those scoring above the mean, and ‘negative’ for those scoring below the mean and level of practice was defined as ‘safe’ for those scoring higher than the median and ‘risky’ for those scoring below the median.\n\nAll questionnaire data was checked, coded, rechecked and entered into Microsoft Excel (v2017), before being checked and double entered into Epi-Info (version 3.5.4). Missing data was removed from analysis by making listwise data deletion. The data were then transformed by converting to true values for terms that have a negative meaning and all data were analyzed using Stata (v 13.0). Descriptive statistics were used to describe demographic characteristics and KAPs about HIV/AIDS. Pearson's chi-squared test and binary logistic regression was used to test the association between factors.\n\nThis research proposal was reviewed and approved by the Mahasarakham University Ethics Committee for Human Research (No.056/ 2561). Permission to collect data in the local area was obtained from the head of the Public Health Provincial Office (Kham Mouane province) in Laos.\n\n\nResults\n\nOf the 939 participants, 61.6% were women and the mean age was 20.7 years (range 18–28 years). Accounting (20.7%), IT (18.3%), and electrical (15.5%) were the major fields of study, and 49% were first year students. In total, 37.4% lived in their family home, 42.1% in private dormitories and 20.5% in a relative’s house or with friends. For monthly income given to the participants from their parents, 57.1% received more than 400,000 Lao Kip (LAK; USD $46.59), 38.3% received 100,000–400,000 LAK (USD $11.65–46.59) and 4.6% received less than 100,000 LAK (USD $11.65). The majority of the money was spent on fees and food, with one fifth spent on alcohol. More than 80% reported drinking alcohol; 54% reported drinking only sips, 31.9% drinking until slightly intoxicated and 13.9% drinking until drunk. Of those that drank alcohol, 50% drank up to 1–2 times a month, 30% drank 1–2 times a week, and 10% drank every day. Free time was spent in employment (44%) and various entertainment; for example, television (44.1%), partying (37.7%), social media (22.4%), games (20%), and reading (30%). Table 1 presents this information.\n\nAlthough the college had never conducted specific HIV/AIDS intervention education, participants had heard of HIV/AIDS and some students reported, to the researchers in person, their peers previously dying of AIDS. Information about HIV came from various sources, such as the internet (76.6%), television (55.3%) or health personnel/teacher (see Table 2).\n\nOverall, the reported level of HIV/AIDS knowledge was low, with very poor/erroneous knowledge of HIV transmission routes: 22.8% correctly reported that HIV transmission could be controlled by having a single partner and using condoms while having sex; 27.9% indicated that HIV could not be transmitted through a mosquito bite; 27.2% incorrectly perceived that couples who are already infected with AIDS did not need to use condoms; 39.4% incorrectly perceived that HIV can be transmitted through sharing a toilet with a PLHIV and almost half (48.9%) incorrectly believed drinking alcohol could disinfect them from HIV infection (Table 3).\n\nLevel was defined as ‘‘Low’’ for scores of less than 60%, ‘medium’ between 60–80% and ‘high’ over 80 [Bloom et al., 1971]\n\nOverall, the level of attitude towards PLHIV was moderate (Table 4). There were many negative stigma issues: more than 80% believed that people who were HIV positive should not reveal themselves to society; and not be allowed to continue his/her teaching or studies. Between 60–70% believed that: requesting a boyfriend to use a condom indicated mistrust; and if someone has AIDS, he/she needn’t maintain their health because it is an incurable disease. In addition, 50–59% felt that: people should not buy from a shopkeeper or food seller who is HIV positive; buying condoms was disgusting and embarrassing; relatives who had progressed to the AIDS stage should be separated from their families due to the disgust of other people; sex education is shameful and shouldn’t be in school programs as it does not need to be taught or instructed; and HIV testing is not necessary as it does not cure disease. Finally, 50.8% believed that healthy-looking men are unlikely to have HIV, so if they are having sex, they shouldn’t have to wear condoms. Table 4 presents fuller details.\n\na Level of attitude was defined as ‘low’ when mean score less than 2.7, ‘medium’ when mean score was between 2.71-3.7 and ‘high’ when mean score was over 3.71 [Likert, 1961]\n\nTable 5 reveals that 60.2% reported behaviors to prevent HIV/AIDS through avoidance of risky behaviors such as: using condoms regularly during sex with casual partners; 75.2% don’t co-habit with boy/girlfriends, which can increase opportunity for sex or watching porn videos; 77% don’t share needles and syringes; 61% don’t have sex with girl/boyfriends who they are just dating; and 52.3% don’t have sex when under the influence of alcohol. When visiting nightclubs, 73.4% of them reported drinking alcohol.\n\ncLevel of practice was also defined as ‘safe’ for those scoring mean over 4.2), ‘risky’ for those scoring mean less than 4.2 (Best, 1981)\n\nThe binary logistic regression reveals that participants with low levels of knowledge were likely to have proportionately higher negative attitudes (OR=1.80; CI=1.07, 1.89; P value <0.001) and risky practices relating to HIV/ AIDS (OR=1.51; CI: 1.07, 1.89; P value=0.014). Demographic factors associated with attitudes and practices include: aged less than 20.6 years was related more significantly to negative attitudes (OR=0.73; CI: 0.56, 0.94; P value=0.016 and OR=0.69; CI: 0.52, 0.91; P value 0.009); being male was associated with proportionately higher risk behaviors (OR=2.27; CI: 1.71, 3.02; P value <0.001); and there was a non-significant association between alcohol drinking and negative attitudes and risky behaviors. Table 6 presents fuller details.\n\na Level of knowledge (K) was defined as ‘low’ for mean scores for less than 50% correct and ‘high’ for over 50% correct. b Level of attitude (A) was defined as ‘positive’ for those scoring >=mean, and ‘negative’ for those scoring <mean. c Level of practice (P) was defined as ‘safe’ for those scoring >=median and ‘risky’ for those scoring <median. d OR=Odds ratio. e CI=Confidence interval of OR. f Significance statistic <=0.05. * Fisher’s exact test.\n\nNote: n=857 for ‘Attitude’ and ‘Gender’ as some participants did not include a response to these items.\n\n\nDiscussion\n\nThis study investigated the association between KAPs and risk factors relating to HIV/AIDs among Kham Mouane Technical-Vocation College students in Lao PDR. Kham Mouane has lower HIV rates than other Lao PDR provinces [Lao Statistics Bureau, 2018]; however, it is slightly increasing among adolescents, with several likely enabling factors. Namely, the college is the only major school in the province with young students, is located in an urban setting and nearly 50% live in private dormitories with restaurants, liquor stores and entertainment venues nearby, increasing opportunities to engage in risky behavior. Furthermore, it is close to cross-border areas like the Mukdahan Province of Thailand, Cambodia and China, which have higher HIV-prevalence [Phimphachanh & Sayabounthavong, 2004]. Furthermore, a key point is that alcohol consumption is associated with increased chances of having unprotected risky sex (AIDsInfo, 2018). We found that more than 80% of the students drink alcohol, half of them drink monthly, 13% drink until drunk and 30% drink until slightly intoxicated. Furthermore, there was no HIV prevention program in their school or community, but it was reported accessible from social media, television or friends by more than 50% of the students.\n\nTo the best of our knowledge, this is the first known study of its type in a vocational technical college in Lao PDR, though it has some similarities with other studies on KAP and HIV prevention among Lao PDR high school students [Boonluane et al.; Kosay et al., 2008] and the Lao PDR survey of risk behaviors in Kham Muane Province [Lao Statistics Bureau, 2018]. There were some differences: 1) overall, these studies [Hansson et al., 2008; Koksal et al., 2005; Mansoor et al., 2008; Tan et al., 2007; Thanavanh et al., 2013] found moderate and high levels of knowledge and attitudes relating to HIV and risky behaviors, whereas our study found low knowledge, and moderate attitude levels but higher safe levels of HIV prevention practice. However, we found concerning amounts of incorrect knowledge, namely; there were misconceptions about HIV transmission, for example, beliefs that HIV can be transmitted through a mosquito bite, or sharing a toilet with an HIV positive person and, significantly, half believed that ‘Alcohol can disinfect HIV’. This indicates that students need much more information and education about the true routes of HIV transmission. About 64.6% of participants displayed more negative attitudes than high school students [Hansson et al., 2008; Koksal et al., 2005; Kosay et al., 2008; Lao PDR Progress Report, 2016; Mansoor et al.,2008; Tan et al., 2007; Thanavanh et al., 2013]. They mainly believed that PLHIV should not be allowed to continue working /studying or cohabiting in their community, because of fear of catching the disease. They believed it was shameful to buy or carry a condom, and it suggests mistrust if a woman requests that her partner uses a condom during sex. Regarding HIV/AIDs prevention behaviors, although the results of this study do not agree with other studies [Hansson et al., 2008; Koksal et al., 2005; Mansoor et al.,2008; Tan et al., 2007; Thanavanh et al., 2013], there were some similarities between this study and Thanavanh et al. (2013). Namely, the percentage of those who drink alcohol (81.4% in the current study versus 91.5%), the percentage of those having sex when under the influence of alcohol (47.4% versus 30.1%), and the percentage of those using condoms when having sex (39.8% versus 43.9%). In the current study, more than 60% of participants were female and there was a higher risk of unsafe sex after drinking alcohol and more misconceptions about condom usage.\n\nThese results suggest the need to for effective participatory education strategies within students’ networks (family, school/ college, public sector, media organizations etc.) to develop proficient and effective HIV awareness, knowledge and practice programs [Kosay et al., 2008; Thanavanh et al., 2013; UNAIDS: PDR Progress report, 2016]. These should involve suitable media sources of information, voluntary counseling, extensive health care, prevention programs, treatment and rehabilitation services, strengthened surveillance capacity and reduction of social stigmatization. Finally, health worker community networks should research and evaluate intervention programs to inform future HIV policy.\n\nThere were some limitations of the study. The study was restricted to only one province, which might limit the generalizability of the findings to other provinces with different contexts. Despite this limitation, we believe these findings offer useful information for researchers and policymakers.\n\n\nConclusion\n\nIn summary, the students surveyed in this Lao PDR Technical-Vocation College had moderate levels of knowledge about and attitudes towards HIV/AIDS prevention. However, misconceptions about HIV transmission, discriminatory attitudes and stigma towards PLHIV were particularly noticeable. Although, most of them had safe HIV behaviors, they also had risky practices, particularly around drinking alcohol, associated with increased likelihood of unsafe sex. Therefore, we recommend that public sector and community networks need to address HIV/AIDS-related education programs with specific interventions for these college contexts to direct practices, knowledge and attitudes in a positive direction and contribute to substantial improvements in HIV/AIDS prevention.\n\n\nData availability\n\nRaw datasets have not been made available at the request of the Mahasarakham University Ethics Committee in order to maintain participant confidentiality. Access to the raw data can be obtained upon request after seeking permission from the Mahasarakham University Ethics Committee. Anyone wishing to access the data should first contact the corresponding author, who will facilitate contact with the Ethics Committee (contact email: research@msu.ac.th). Access will be granted under the conditions that the person or organization seeking access provides sufficient rationale, highlighting the benefits for use of this data, and are operating under appropriate research ethics.\n\nFigshare: Knowledge, attitude and prevention behavior related to HIV/AIDS among students of a college in Lao-PDR: a cross-sectional study, https://doi.org/10.6084/m9.figshare.11846097.v1 [Wongkongdech, 2020].\n\nThis project contains the following extended data:\n\n- Questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe acknowledge the contribution and cooperation of the all participating students and teachers of Kham Mouane Technical-Vocation College who made the study possible. We also acknowledge all directors and staff of provincial health departments and health sector for their cooperation and all expert from Kham Mouane Hospital who checked content validity. We also thank Professor John F Smith for editorial and English presentation feedback on the manuscript.\n\n\nReferences\n\nAIDsInfo: HIV and Drug and Alcohol Users. 2018; [Cited 2019 March26]. Reference Source\n\nBest JW: Research in Education, 4 th ed. New Jersey: Prentice – Hall Inc. 1981. Reference Source\n\nBloom BS, Hastings JT, Madaus GF: Handbook on formative and summative evaluation of student learning. 1971. Reference Source\n\nBowring AL, Pasomsouk N, Higgs P, et al.: Factors Influencing Access to Sexual Health Care Among Behaviorally Bisexual Men in Vientiane, Laos: A Qualitative Exploration. Asia Pac J Public Health. 2015; 27(8): 820–34. PubMed Abstract | Publisher Full Text\n\nHansson M, Stockfelt L, Urazalin M, et al.: HIV/AIDS awareness and risk behavior among students in Semey, Kazakhstan: a cross-sectional survey. BMC Int Health Hum Rights. 2008; 8: 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHIV and AIDs data HUB for Asia Pacific Evidence to Action: LAO PDR: Key Fact for HIV. 2018; [Cited 2018 Febuary14]. Reference Source\n\nKoksal S, Namal N, Vehid S, et al.: Knowledge and attitude towards HIV/AIDS among Turkish students. Infect Dis J Pakistan. 2005; 14: 118. Reference Source\n\nKosay HO, Jutatip SI, Boonyong KI: Safe sex intention toward HIV/AIDS Prevention among high school student of Khammouane province, LAO P.D.R. J Public Health Dev. 2008; (6): 79–86. Reference Source\n\nLao Statistics Bureau: Lao Social Indicator Survey II 2017. Survey Findings Report. Vientiane, Lao PDR: Lao Statistics Bureau and UNICEF. 2018. Reference Source\n\nLikert RA: New Patterns of Management. New York: McGraw-Hill Book Company Inc. 1961. Reference Source\n\nMansoor AB, Fungladda W, Kaewkungwal J, et al.: Gender differences in KAP related to HIV/AIDS among freshmen in Afghan universities. Southeast Asian J Trop Med Public Health. 2008; 39(3): 404–18. PubMed Abstract\n\nMayuree I, Pannee B: Application of Health Belief Model and Self-Eefficacy for Preventive Behavior of AIDs in Secondary School in Chaiyaphum Province. Journal of Health Science Research. 2015; 8(2): 25–32.\n\nPhimphachanh C, Sayabounthavong K: The HIV/AIDS/STI situation in Lao People's Democratic Republic. AIDS Educ Prev. 2004; 16(3 Suppl A): 91–99. PubMed Abstract | Publisher Full Text\n\nPhrasisombath K, Thomsen S, Hagberg J, et al.: Knowledge about sexually transmitted infections (STIs) and attitudes toward female sex workers with STI symptoms among health care providers in Laos. Asia Pac J Public Health. 2012; 24(6): 940–52. PubMed Abstract | Publisher Full Text\n\nSilakoune T: World AIDS Day 2017 promotes “Right to Health”. 2017; [Cited 2018 August5]. Reference Source\n\nSychareun V, Thomsen S, Chaleunvong K, et al.: Risk perceptions of STIs/HIV and sexual risk behaviours among sexually experienced adolescents in the northern part of Lao PDR. BMC Public Health. 2013; 13: 1126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan X, Pan J, Zhou D, et al.: HIV/AIDS knowledge, attitudes and behaviors assessment of Chinese students: a questionnaire study. Int J Environ Res Public Health. 2007; 4(3): 248–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThanavanh B, Harun-Or-Rashid M, Kasuya H, et al.: Knowledge, attitudes and practices regarding HIV/AIDS among male high school students in Lao People's Democratic Republic. J Int AIDS Soc. 2013; 16(1): 17387. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUNAIDS: Country factsheets of Lao People’s Democratic Republic 2017. [Cited 2018 January14]. Reference Source\n\nUNAIDS: UNAIDS Data 2018. 2018; [Cited 2018 Febuary12]. Reference Source\n\nUNFPA: Lao Social Indicator Survey II 2017-18. 2017; [Cited 2018 ctober5]. Reference Source\n\nWongkongdech R: Knowledge, attitude and prevention behavior related to HIV/AIDS among students of a college in Lao-PDR: a cross-sectional study. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.11846097.v1\n\nWorld Health Organization: A month-long World AIDS Day celebration in Lao People’s Democratic Republic. 2019; [Cited 2019 Febuary1]. Reference Source"
}
|
[
{
"id": "61203",
"date": "23 Mar 2020",
"name": "Roger Chun-Man Ho",
"expertise": [
"Reviewer Expertise Depression",
"HIV",
"meta-analysis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study studies knowledge, attitude and prevention behavior related to HIV/AIDS among college students in Lao and there are a number of misunderstandings by respondents. I have the following recommendations:\nUnder the methods, the authors need to explain how they got a 95% CI of the population. There must be a measurement outcome. 750 is one quarter of 2734, how did the authors get 95% CI? The authors need to explain more.\n\nThe authors should compare their findings with other ASEAN countries. Please add the following statement at the end of the first paragraph:\n“.... more than 50% of the students. In this study, only 22.8% respondents correctly indicated that using condoms while having sex. This figure is lower than in other ASEAN countries. In contrast, 48.1% of young Vietnamese used condoms at their latest sexual intercourse (Ngoc et al., 20201).”\n\nThe authors should mention education strategies in other ASEAN countries. Please add the following statement:\n“Other ASEAN countries adopted mobile health apps or interventions for disease prevention (Do et al., 20182). This strategy could be feasible to proliferate the benefits of HIV prevention in youths and adolescents in Laos.”\n\nPlease add one additional limitation:\n“.... other provinces with different contexts. This study did not explore other knowledge on HIV which was assessed in other ASEAN countries. For example, 98.2% of Vietnamese believed that HIV could be transmitted from mother to child (Hoang et al., 20193) but it would be important to assess the knowledge of young people in Lao.”\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": "68031",
"date": "20 Aug 2020",
"name": "Ivan Marbaniang",
"expertise": [
"Reviewer Expertise Mental health",
"HIV",
"sexual minorities",
"health services",
"adolescent mental health",
"Epidemiology",
"Causal analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI commend the authors for their work, which remains a crucial area that has not been fully addressed in South East Asia, sparing a few countries.\n\nThere are however a number of methodological issues and issues with interpretation which makes these findings less valid.\n\nThe authors mention that, \"A list of 2,734 student names was obtained and of these, participants were selected by simple random sampling (939 names were drawn), ensuring that the same proportion of each academic year was invited.\"\n\nThis statement is not clear, do the authors mean an equal proportion of students from each year (which is clearly not true, since 49% of the participants belong to Grade I) or do they mean an equal proportion relative to the number of students in each Grade?\n\nIt is also unclear if the categorization of low, medium, high based for both attitude and risky behavior was based off cut-offs specified by Likert and Best? If so, do the authors think that these are appropriate for their setting?\n\nThe authors change the definition of low knowledge from 60% to 50% when they use it in the logistic regression model, what is the justification for this?\n\nDid the authors consider using an ordinal logistic regression so that they did not need to change their definitions? Please see the link for ordered logistic regression below: https://stats.idre.ucla.edu/stata/dae/ordered-logistic-regression/\n\nWhy did the authors use the mean to describe a cut-off for the attitude, but the median to describe a cut-off for risky behavior? How robust are the authors' estimates to the specification of these cut-offs? Did they consider conducting any sensitivity analyses?\n\nIn the Results section, it is unclear if the authors are describing practices as practiced by these students (as the subheading indicates in the Results section) or knowledge about these practices (as indicated by Table 5)? The authors switch between the two and what they intend to describe becomes ambiguous.\nMoreover, in the results there are a number of inconsistencies which the authors have not explained:\nExample 1: \"60.2% reported behaviors to prevent HIV/AIDS through avoidance of risky behaviors such as: using condoms regularly during sex with casual partners\" AND \"61% don’t have sex with girl/boyfriends who they are just dating\".\nSince people can use condoms only when having sex (conventionally), if 61% of them are not having sex then it is not possible that 60.2% of them are using a condom.\n\nExample 2: \"52.3% don’t have sex when under the influence of alcohol\" AND \"73.4% of them reported drinking alcohol\"\nThese statements taken together would imply, contrary to what the authors state in their discussion, that alcohol may cause risky sexual behavior. In fact these two statements taken together would imply that students were less likely to indulge in high risk behavior after consuming alcohol.\n\nI would suggest that the authors try stratifying their analysis by gender, to see if they see a heterogeneity of effects based on gender.\n\nThe authors should consider using a language editing service, if it is available to them to make their language a lot more clearer.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-181
|
https://f1000research.com/articles/8-1983/v1
|
25 Nov 19
|
{
"type": "Method Article",
"title": "Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice",
"authors": [
"Christopher Blackwood"
],
"abstract": "Background: The neurosphere assay is a powerful tool to study neural stem cell biology. The objective of this protocol is to create a simple and rapid approach to generate neurospheres from the dorsal lateral ganglionic eminence of late embryonic (day 17) mice. This method predicts the average number of neurospheres and provides an approximation of its expected size after 7 days in vitro. Characterization of numbers and sizes will provide investigators with quantitative data to advise on the implementation of downstream applications, including immnocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro. Furthermore, we found this method yields different sizes of neurospheres. Lastly, using an anti-GFAP antibody, we confirm that these neurospheres can be used for immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of neurospheres that will be useful for further advances in the area of stem cell biology.",
"keywords": [
"Embryonic",
"mechanical dissociation",
"neurosphere",
"neural stem cell",
"progenitor cell"
],
"content": "Introduction\n\nNeural stem cells are a tissue-specific subtype of self-renewing and multipotent cells that will produce several mature cell types. The neurosphere assay is an important tool that has been extensively employed to study neural stem cell biology1. Since its introduction some 25 years ago2, neurospheres have been used to study neurogenesis3, genes that regulate self-renewal4, and molecular mechanisms that control neuronal and glial differentiation3,5–8. Although there are many neurosphere protocols, the expected number and size of neurospheres generated after a week in vivo is not fully understood.\n\nWe developed a simple dissection technique that helps to maximize the number of neurospheres that can be produced in culture. Furthermore, we characterize the expected sizes of neurospheres after a week in vitro. With some other techniques, a brain slicer or other means are used to obtain thick slices of brain tissue from late embryonic stages9,10. The area surrounding the ventricle is then microdissected from a given slice to enrich for neural stem/progenitor cells. This approach, while effective, can be painstaking and may require expensive specialized equipment. In our approach, no specialized equipment is needed. The lateral ventricle is visualized with a stereomicroscope, and the surrounding tissue is simply trimmed away using a razor blade or scalpel. This method requires only half of a single brain, and generates reproducible numbers of neurospheres after a week. Our method also provides an approximation of the expected sizes for neurospheres after a week in vitro.\n\n\nMethods\n\nThe animals were housed in the AAALAC-accredited East Campus Research Facility and Transgenic Mouse Core Facility in the Veterinary College of Cornell University. All animal procedures were performed in accordance with the guidelines outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, eighth Edition. Animals were approved by Cornell University’s Animal Care and Use Committee (IACUC; #01-75). Mice were maintained on a mixed 129Sv/C57BL/6 background and housed on a reverse light-dark cycle. Food and water were continuously available. Male and female mice mated overnight. The following morning females were separated and checked for vaginal plug. Pregnant mice were euthanized using CO2 asphyxiation followed by cervical dislocation consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and the Cornell IACUC. Embryonic day 17.5 (E17.5; E0.5 was defined as the first detection of the vaginal plug) male and female embryos were dissected. A total number of n=5 mice were used for primary neurospheres experiments.\n\nThe mouse brain was sliced down the center into two hemispheres. Using one hemisphere, placed sagittal orientation, dissect out the dorsal lateral ventricle (see Figure 1). The dissections were guided using stereotaxic coordinates (A/P 1 mm, M/L 1 mm, D/V 2.3 mm) from Paxinos and Franklin (2007) atlas source.\n\nTissue from the dLGE is dissected and dissociated. Single cells are grown in media containing EGF and B27. After 7 days in vitro neurospheres are approximately 30 μm to 250 μm in size. Neurospheres generated can be used for various downstream applications.\n\nNeurospheres were fixed with 4% paraformaldehyde, and blocked in goat serum containing 0.5% Triton. Immunofluorescence analysis of protein expression was performed using rabbit anti-Glial Fibrillary Acidic Protein (GFAP) Antibody (Millipore; RRID:AB_2109645; ab5804; 1:100). Secondary antibodies used were biotinylated goat anti-rabbit (Abcam; RRID:AB_2661852; ab64256; 1:1000) and streptavidin alexa fluor 488 conjugate (ThermoFisher Scientific; RRID:AB_2315383; S11223; 1:500).\n\nImages were taken with a Canon EOS Rebel XS. The field of view represent a length of 783 μm and a width of 522 μm. Data is derived from single random pictures of each well. Per animal, 3-4 wells were analyzed. A total of 5 individual animal were analyzed. Size measurements and neurosphere counts were analyzed in Adobe Photoshop (version 10.0.1) CS3 Extended (Adobe; San Jose, CA) using the measurement function (ImageJ is an open-access alternative that can be used to perform this function). Statistical analyses were carried out as previously described11,12. Briefly, data were analyzed using PRISM 8 (version 8.3.0) (GraphPad Software; San Diego, CA) by performing one-way ANOVA. If the main effect was significant (p < 0.05), Bonferroni’s multiple comparision post hoc test were used to compare the different replicates. The null hypothesis was rejected at p < 0.05. Data is made available on figshare open access platform (Metrics of Primary Neurospheres). Error bars represent standard error of the mean (±SEM).\n\nThis protocol is designed to generate neurospheres from a single embryo. Multiply all values as needed to generate neurospheres from additional embryos. See Table 1 and Table 2 for premade solutions and materials needed.\n\n1. Set-up prior to tissue dissection\n\n1.1) Establish breeding pairs of mice to obtain embryonic day 17 (E17) embryos. Day 0 is defined as the day a vaginal plug is detected.\n\n1.2) Prepare sterile surgical tools (scissors for decapitation, #5 forceps, razor blades).\n\n1.3) Add 20 mls of Hank’s buffer to each of two 10 cm petri plates and place on ice. Add 5 mls Hank’s buffer to a 15 ml tube and also place on ice. Reserve another 50 mls of room temperature Hank’s buffer.\n\n1.4) Prewarm 10 mls of Hank’s-low BSA at 37°C\n\n1.5) Prewarm 5 mls of Hank’s-high BSA at 37°C.\n\n1.6) Prewarm 10 mls of DMEM/F12 with serum at 37°C.\n\n1.7) Prewarm 5–15 mls of neurosphere media at 37°C. Amount is based on number of desired wells.\n\n1.8) Prewarm 2 mls of 0.25% trypsin/EDTA at 37°C.\n\nNote: Breeding and euthanasia of all animals should be performed in accordance with an institutionally approved animal care and use protocol.\n\n2. Tissue dissection\n\n2.1) Spray the abdomen with 70% ethanol, and make an incision to expose the uterus. Remove the uterus and transfer it to an empty petri plate.\n\n2.2) Remove embryos from the uterus, spray desired number with 70% ethanol, and decapitate one or more embryos. Rinse each decapitated head in one petri plate containing ice-cold Hank’s buffer, and then transfer to the second petri plate containing Hank’s buffer on ice.\n\n2.3) Use forceps to remove the skin and skull. Remove the brain and place in an empty petri dish.\n\n2.4) Separate the two hemispheres with a razor blade, and place one half of a brain on its lateral surface.\n\n2.5) Using a stereomicroscope, identify the location of the lateral ventricle on the medial surface (the dorsal region of the lateral ventricle contains the dLGE). The ventricle is visible as a T-shaped structure that is slightly darker than the rest of the brain. Using a razor blade or a scalpel, sequentially trim away the brain surrounding the ventricle on all four sides.\n\n2.6) Transfer the dissected tissue into the 15 ml tube of Hank’s buffer on ice.\n\n2.7) If neurospheres are to be isolated from additional embryos (e.g. because of low yield), keep tube on ice until all dissections are complete.\n\n3. Primary neurosphere culture\n\n3.1) Spin sample at 300 RCF in a clinical centrifuge for 3 min. to pellet tissue.\n\n3.2) Aspirate off the supernatant and add 2 mls of pre-warmed trypsin/EDTA. Incubate at 37°C for 15 min. with intermittent swirling.\n\n3.3) Spin tube at 300 RCF for 2 min.\n\n3.4) Add 10 mls of room temperature Hank’s to trypsin/tissue mixture and incubate at 37°C for 5 min. with intermittent swirling. Spin culture at 300 RCF for 3 min. and remove the supernatant.\n\n3.5) Repeat wash step 3.4 two additional times.\n\n3.6) Aspirate the supernatant and add 4 mls of Hank’s-low BSA.\n\n3.7) Triturate the tissue gently and slowly approximately 10 times with an 18-gauge needle until tissue chunks appear relatively uniform in size. Avoid creating bubbles or foam.\n\n3.8) Triturate the crude cell suspension gently and slowly approximately 7–10 times with a 21-gauge needle until tissue chunks appear relatively uniform in size.\n\n3.9) Triturate the suspension approximately 4–5 times with a 23-gauge needle until suspension appears uniform.\n\n3.10) Add 3 mls of Hank’s-high BSA solution to a 15 ml tube. Slowly add the cell suspension to the bottom of the tube underneath the Hank’s-high BSA solution using a 23-gauge needle.\n\n3.11) Centrifuge at 300 RCF for 5 min.\n\n3.12) Aspirate supernatant and resuspend cells with 3 mls of prewarmed Hank’s-low BSA.\n\n3.13) Centrifuge at 300 RCF for 5 min.\n\n3.14) Aspirate supernatant, and resuspend cells in 5 mls of prewarmed DMEM/F12 with serum.\n\n3.15) Incubate tubes for 2–4 hours at 37°C to reduce bacterial contamination.\n\n3.16) Centrifuge at 300 RCF for 5 min.\n\n3.17) Resuspend cells in 1 ml of prewarmed neurosphere media.\n\n3.18) Count cells with a hemocytometer. Plate 10,000 cells in a volume of 250 μl in each well of a 48-well plate. Plate at least 8 wells to ensure adequate numbers of neurospheres.\n\n3.19) Incubate at 37°C in a humidified incubator with 5% CO2.\n\n3.20) Neurospheres should form within 3–4 days. At day 3, add an additional 100 μl of neurosphere media to each well.\n\nThe extended version of our protocol can be used to obtain secondary neurospheres (https://dx.doi.org/10.17504/protocols.io.823hygn).\n\n\nResults\n\nFigure 1 shows a general overview of the neurospheres assay. A picture representation of primary neurospheres grown for 7 days in vitro is given in Figure 2A. The statistical analysis using a one-way ANOVA revealed no significant difference between the average numbers of neurosphere per field of view (F(4,13) = 0.666; p = 0.6268; N=5) (Figure 2B).\n\n(A) Average size of primary neurospheres per field of view after 7 days in vitro. (B) Average number of neurospheres per field of view after 7 days in vitro (N=5). Scale bar = 100 μm.\n\nFigure 3 shows in the variation in sizes of neurospheres grown for 7 days in vitro (Figure 3A). The statistical analysis using one-way ANOVA revealed a significant difference in the sizes of neurospheres between the replicates (F(4,129) = 11.666; p < 0.0001) (Figure 3A). Using similar analyses, we found significant differences between the size classification of neurospheres that were less than 50 μm, between 50-100 μm, and greater than 100 μm (F(2,379) = 424; p < 0.0001) (Figure 3B). Post hoc analysis using Bonferroni’s multiple comparison revealed a significant difference between the primary neurospheres that were greater than 100 μm compared to neurospheres that were less than 50 μm (p <0.0001) or between 50-100 μm (p <0.0001) (Figure 3B). Similarly, we found a substantial difference between primary neurospheres that were greater than 100 μm compared to neurospheres that were between 50 -100 μm (p <0.0001) (Figure 3B). Numbers and sizes of neurospheres, alongside the raw images used to produce these values, are available as Underlying data13.\n\n(A) Average size of primary neurospheres per field of view after 7 days in vitro (N=5). (B) The comparison of the numbers of neurosphere that are less than 50 μm, between 50-100 μm, and greater than 100 μm. Key to statistics **, *** = p <0.01, 0.001, respectively, in comparison to NS less than 50 μm or replicate 1. #, ##, ###, = p < 0.05, 0.01, 0.001, respectively, in comparison to NS between 50-100 μm or replicate 2. $ = p <0.05 in comparison to replicate 3 (minimum of 5 independent samples; N=29, N=214, N=136, respectively to NS<50 μm, NS between 50 μm -100μm, NS>100 μm).\n\nNeurospheres can be used for a variety of purposes, including immunocytochemistry. Figure 4 is a picture of a primary neurosphere immunostained using an anti-GFAP (Figure 4A; arrowhead) antibody and counterstained with DAPI (Figure B; arrowhead).\n\nVisual representation of immunocytochemistry staining of neurospheres using (A) anti-GFAP antibody (green) and (B) counterstained with DAPI (blue) after 7 days in vitro. Arrowheads represent positive GFAP signal. Scale bar = 100 μm.\n\n\nDiscussion\n\nThere are several key steps that are important to maximize the yield and health of neurospheres. The most important step is to incubate the final triturated culture in the prewarmed DMEM/F12 with serum for 2–4 hours. This incubation time is necessary in order for the antibiotics in the media to inhibit growth of bacteria, and reduce subsequent contamination. Another essential step is to perform the trituration as gently as possible. Over-trituration, or trituration with great force, will result in increased cell death.\n\nAdditional modifications to the approach may be required. We have provided general guidelines for the number of cells to be plated per well and the expected number of neurospheres per field of view. If too many cells are plated per well, differentiation may occur. If so, either reduce the number of cells plated per well or increase the concentration of EGF to 20 ng/ml. If desired, trypsin inhibitor can be substituted for the FBS during the generation of primary neurospheres to inactivate proteolytic activity.\n\nOur approach utilizes a mechanical trituration to dissociate cells. By sequentially processing the cells through successively higher gauge needles, we obtain very few clumps of cells. This eliminates the need for a cell strainer, which can reduce yield, as well as the time needed to perform enzymatic dissociation. Although cell death can be increased with mechanical dissociation, in our hands, this does not seem to affect neurosphere yield or health.\n\nOne advantage of our approach is the ease with which tissue surrounding the lateral ventricle can be isolated from the rest of the brain. Although this dissection is relatively crude, it is easier and faster than other approaches, which may require a brain slicer and/or microdissection9,10. This method eliminates the need for specialized equipment while generating high numbers of neurospheres from a single brain. This method has been shown in other studies to generate neurospheres greater than 300 μm in size when grown for 12 days in vitro3.\n\n\nData availability\n\nFigshare: Metrics of Primary Neurospheres.xlsx. https://doi.org/10.6084/m9.figshare.10280288.v113.\n\nThis project contains the following underlying data:\n\nSpreadsheet containing numbers and sizes per field of view.xlsx (details on numbers and sizes of neurospeheres produced from each mouse).\n\nAll raw, unprocessed microscope images used to produce results.\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 Michael T. McCoy and NCI editorial review board for their careful review of the manuscript. Additionally, we thank David M. Lin for his assistance, providing space, and editorial support, reagent, and equipment. Lastly, we thank the reviewers for their valuable feedback.\n\n\nReferences\n\nCampos LS: Neurospheres: insights into neural stem cell biology. J Neurosci Res. 2004; 78(6): 761–9. PubMed Abstract | Publisher Full Text\n\nReynolds BA, Weiss S: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992; 255(5052): 1707–10. PubMed Abstract | Publisher Full Text\n\nBlackwood CA: Jagged1 is Essential for Radial Glial Maintenance in the Cortical Proliferative Zone. Neuroscience. 2019; 413: 230–238. PubMed Abstract | Publisher Full Text\n\nNyfeler Y, Kirch RD, Mantei N, et al.: Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal. EMBO J. 2005; 24(19): 3504–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen X, Chen L, Zhang R, et al.: ADAM17 regulates self-renewal and differentiation of U87 glioblastoma stem cells. Neurosci Lett. 2013; 537: 44–9. PubMed Abstract | Publisher Full Text\n\nJensen JB, Bjorklund A, Parmar M: Striatal neuron differentiation from neurosphere-expanded progenitors depends on Gsh2 expression. J Neurosci. 2004; 24(31): 6958–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKong SY, Park MH, Lee M, et al.: Kuwanon V inhibits proliferation, promotes cell survival and increases neurogenesis of neural stem cells. PLoS One. 2015; 10(2): e0118188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanchez-Mendoza EH, Schlechter J, Hermann DM, et al.: Characterization of Seeding Conditions for Studies on Differentiation Patterns of Subventricular Zone Derived Neurospheres. Front Cell Neurosci. 2016; 10: 55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDizon M, Szele F, Kessler JA: Hypoxia-ischemia induces an endogenous reparative response by local neural progenitors in the postnatal mouse telencephalon. Dev Neurosci. 2010; 32(3): 173–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLouis SA, Mak CK, Reynolds BA: Methods to culture, differentiate, and characterize neural stem cells from the adult and embryonic mouse central nervous system. Methods Mol Biol. 2013; 946: 479–506. PubMed Abstract | Publisher Full Text\n\nBlackwood CA, Hoerle R, Leary M, et al.: Molecular Adaptations in the Rat Dorsal Striatum and Hippocampus Following Abstinence-Induced Incubation of Drug Seeking After Escalated Oxycodone Self-Administration. Mol Neurobiol. 2019; 56(5): 3603–3615. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlackwood CA, Leary M, Salisbury A, et al.: Escalated Oxycodone Self-Administration Causes Differential Striatal mRNA Expression of FGFs and IEGs Following Abstinence-Associated Incubation of Oxycodone Craving. Neuroscience. 2019; 415: 173–183. PubMed Abstract | Publisher Full Text\n\nBlackwood CA: Metrics of Primary Neurospheres.xlsx. figshare. Online resource. http://www.doi.org/10.6084/m9.figshare.10280288.v1"
}
|
[
{
"id": "57031",
"date": "19 Dec 2019",
"name": "M.L. Shawn Bates",
"expertise": [
"Reviewer Expertise My area of expertise is behavioral neuroscience",
"including in vivo electrophysiology",
"and various learning and conditioning 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\nThis manuscript describes a protocol that can be used to generate primary neurospheres from the tissue surrounding the lateral ventricle in mice in the late embryonic period. The method is a relatively simple technique that will provide investigators with viable neurospheres that can be used in further studies.\nThe protocol described here is important, and will likely be useful for many researchers who utilize neural stem cells. However, I have some constructive criticism and points that may require clarification, see below:\nHow long does it usually take to generate neurospheres? Is the fact that this protocol takes 7 days the rationale for developing it?\n\nIs there a benefit to generating different sizes of neurospheres?\n\nWhy only use anti-GFAP (in essence, my question is why only visualize glia)?\n\nUnder the “Data acquisition and statistics subsection”:\nThe field of view represents a length of... Bonferroni's multiple comparison post hoc test… A total of 5 individual animals were analyzed…\n\nThe significance of the protocol is somewhat understated. I would add language to the Discussion to highlight the novelty of this approach. I would also include more suggestions for potential ways that others can use the protocol.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "57030",
"date": "24 Dec 2019",
"name": "Oleksii Shandra",
"expertise": [
"Reviewer Expertise Immunohistochemistry",
"glial cells",
"astrogliosis",
"epilepsy",
"traumatic brain injury",
"two photon microscopy",
"neuronal and astrocytic calcium 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\nThis method article written by Dr. Blackwood is aimed to provide a simple and reproducible protocol generating neurospheres from lateral ganglionic eminence in late embryonic mice and demonstrate its applicability for further immunocytochemical assays. Several aspects and protocol steps, however, require clarification:\nThis protocol includes multiple sections with crucial steps. One suggestion that could make the protocol even greater for educational purposes is providing a brief paragraph to sections 1, 2 and 3 (prior to individual steps) that will explain what is important in this section and list all the necessary reagents and tools that will be used. This will help anyone who is using this protocol for the first time to prepare all required items for this protocol section easier rather than finding the specific reagents and volume from each individual step.\n\nTable 1 details equipment, reagents and catalogue numbers; however, the digital camera used for acquiring the images is not listed. The model of stereo stereomicroscope (step 2.5) should be added to table 1, as well as the optimum magnification required or used in this protocol. The type and model of humidified incubator in step 3.19 would also be helpful for reproducibility.\n\nSection 3 called “primary neurosphere culture” is the main section explaining the method of neurosphere generation. In other publications, e.g. Raponi et al. (2007)1, a filtration using a 40um-mesh-nylon cell strainer to dissociate cells after triturating. It may be valuable to add a note after trituration step that at this point similar step could be done if the purpose is to obtain uniform suspension from tissue.\n\nThe “Data acquisition and statistics” section requires more details. Specifically, the additional valuable information could be addressing the distance from the culture plate, whether this is an important factor for further size measurements and if so what was the distance between the camera and the well. What was the zoom factor used here?\n\nThe section “Primary neurospheres at 7 days in vitro can be used for immunocytochemistry” is aimed to demonstrate applicability of this protocol. The GFAP/DAPI is used as an example. While it is stated in the “Introduction” that neurospheres have been used to study molecular mechanisms controlling differentiation of both neural and glial cells, it is not very clear why GFAP was used for this example. Maybe author could clarify or explain why more specific markers were not used here instead of GFAP. From the Fig. 4 it looks as only 1 cell within the neurosphere was GFAP+, hence I believe using a more specific antibody could produce more representative result. Several references provided to this review detail markers that could also be used.\n\nLastly, I believe another interesting question that could be potentially valuable here is the step explaining the reader, which factors or characteristics of the neurosphere help to determine that the neurosphere is mature.\n\nIn summary, after addressing these comments, I believe this method article is a good protocol that can be used as a reference and educational tool for both researchers that are familiar with similar techniques and those who approach it for the first time.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "5290",
"date": "11 Mar 2020",
"name": "Christopher Blackwood",
"role": "Author Response",
"response": "Comment 2.1:This protocol includes multiple sections with crucial steps. One suggestion that could make the protocol even greater for educational purposes is providing a brief paragraph to sections 1, 2 and 3 (prior to individual steps) that will explain what is important in this section and list all the necessary reagents and tools that will be used. This will help anyone who is using this protocol for the first time to prepare all required items for this protocol section easier rather than finding the specific reagents and volume from each individual step.Response 2.1: Thank you for your comments. We have revised accordingly and provided a brief paragraph to section 1, 2, and 3 prior to the individual steps. Section 1:Set-up prior to tissue dissection NOTE: Breeding and euthanasia of all animals should be performed in accordance with an institutionally approved animal care and use protocol. Sterilize all surgical instruments packed in aluminum foil in an autoclave at 121°C (15 psi) for 30mins. This includes a scissors, forceps, and razor blades. Before starting all premade solutions should be warmed to 37°C. Section 2: Tissue dissection NOTE: Make freshly prepared 70% ethanol spray prepared. Have a petri dish (100 mm) prepared with ice-cold Hank’s buffer kept on ice that will be used for embryos after dissection. Afterwards, additional petri dishes will be needed to place in each dissected brain (35 mm) Section 3:Primary neurosphere cultureNOTE: Before starting warmed to 37°C the following solutions: trypsin/EDTA serum media, Hank’s-low, and Hank’s-high. In this section you will need the 18-gauge, 21-gauge, 23-gauge needle will be needed for trituration steps. Trituration should be performed gently and slowly to avoid killing cells. Hemocytometer will be needed to count cells. Comment 2.2 Table 1 details equipment, reagents and catalogue numbers; however, the digital camera used for acquiring the images is not listed. The model of stereo stereomicroscope (step 2.5) should be added to table 1, as well as the optimum magnification required or used in this protocol. The type and model of humidified incubator in step 3.19 would also be helpful for reproducibility. Response 2.2. We have revised the Table 1 to add the Camera and Humidifier. We also included information for the optimum magnification to Data acquisition and statistics section. “Images were taken with a Canon EOS Rebel XS camera. (Canon USA; Melville, NY). The optimum magnification is approximately 5x with 3888 x 2592 dimensions. Camera was connected to the trinocular port of the stereomicroscope (Carl Zeiss Stemi 305; White Plains, NY) using Mount Adaptor EF-EOS (6098B007AA; Canon; Melville, NY). Comment 2.3 Section 3 called “primary neurosphere culture” is the main section explaining the method of neurosphere generation. In other publications, e.g. Raponi et al. (2007)1, a filtration using a 40um-mesh-nylon cell strainer to dissociate cells after triturating. It may be valuable to add a note after trituration step that at this point similar step could be done if the purpose is to obtain uniform suspension from tissue. Response 2.3. Thank you have revised the “primary neurosphere culture” section accordingly. “After trituration, if a uniform suspension has not been achieved an alternative method used in previous protocols are strainers 14.” Comment 2.4 The “Data acquisition and statistics” section requires more details. Specifically, the additional valuable information could be addressing the distance from the culture plate, whether this is an important factor for further size measurements and if so what was the distance between the camera and the well. Response 2.4: We have revised the Data acquisition and statistics” section according to your recommendation. The scale bar should be used to advise the size of the neurosphere. Additionally, we have included information about the working distance to the “Data acquisition and statistics” section. “The working distance was defined as the amount of room required between the top of the neurosphere and the bottom of the objective lens in order for the image to be in focus. The stereomicroscope was used at a working distance of ~110 mm. Due to the variation in neurosphere size, 110 mm should be adjusted to focus on the desire region of the neurosphere to provide optimal focus.” Comment 2.5: The section “Primary neurospheres at 7 days in vitro can be used for immunocytochemistry” is aimed to demonstrate applicability of this protocol. The GFAP/DAPI is used as an example. While it is stated in the “Introduction” that neurospheres have been used to study molecular mechanisms controlling differentiation of both neural and glial cells, it is not very clear why GFAP was used for this example. Maybe author could clarify or explain why more specific markers were not used here instead of GFAP. From the Fig. 4 it looks as only 1 cell within the neurosphere was GFAP+, hence I believe using a more specific antibody could produce more representative result. Several references provided to this review detail markers that could also be used. Response 2.5: To provide more evidence that this protocol can be used to stain neurospheres, we have included an additional picture to the panel (Figure 4C). Larger neurospheres will contain more GFAP-expressing neurons that can be labeled. Furthermore, we have reference our previous published report that demonstrated other immunocytochemistry using other antibodies. Comment 2.6: Lastly, I believe another interesting question that could be potentially valuable here is the step explaining the reader, which factors or characteristics of the neurosphere help to determine that the neurosphere is mature. Response 2.6: Thank you for the recommendation. We have revised the manuscript to expand this point in our discussion. “A sign of bacteria contamination is reduced visibility of the media. Another marker of an unhealthy culture is a large number of differentiated neurons surrounding neurospheres. Indicators of differentiation are the large presence of axons and dendrites in your cultures.This can be caused by depletion of growth factors. If this is the case, we recommend that you increase the concentration of EGF. Another cause of differentiation is too many cells in your prep. This leads to over crowdedness. We recommend splitting the culture to a lower density and decreasing the number of neurons that are plated per 48 well.”"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1983
|
https://f1000research.com/articles/9-175/v1
|
10 Mar 20
|
{
"type": "Research Article",
"title": "Prevalence of sarcopenia in an elderly population in rural South India: a cross-sectional study",
"authors": [
"Nishat Shaikh",
"R Harshitha",
"Madhavi Bhargava",
"Nishat Shaikh",
"R Harshitha"
],
"abstract": "Background: Sarcopenia is a condition common in the elderly characterized by progressive and generalized loss of skeletal muscle mass and strength leading to poor quality of life. According to a working definition proposed by the European Working Group on Sarcopenia in Older People (EWGSOP), the criteria for a diagnosis of sarcopenia is based on documentation of low muscle mass with either poor muscle strength or low physical performance. The objective of the present study was to determine the prevalence of sarcopenia in the rural elderly population of South India. Methods: We conducted a cross-sectional survey of 240 elderly people, 60 years and above, 118 men and 122 women, in rural areas of Dakshina Kannada district. We measured their height, weight, gait speed (using an 8-feet walk test) and muscle strength (using a handgrip dynamometer). Appendicular skeletal mass was calculated using height and weight adjusted for sex and Asian people using the Lee formula. Results: Sarcopenia was found in 34 (14.2%) participants. Of all those having sarcopenia, 27 (79.4%) were ≤75 years, 30 (88.2%) were women, 27 (79.4%) were married, 23 (67.6%) had below poverty line status. Conclusions: The prevalence of sarcopenia found in the present study was 14.2% in an elderly population more than 60 years of age and was found to be more in women. Large-scale multi-centric community-based surveys will help determine the actual burden of sarcopenia in India.",
"keywords": [
"sarcopenia",
"muscle strength",
"muscle function",
"gait speed",
"grip strength",
"elderly"
],
"content": "Introduction\n\nThere has been an increase in longevity globally. According to the United Nations, the proportion of people above the age of 60 years is 13% at a global level and 8.6% in India1,2. Sarcopenia is a condition that is common in elderly people and is characterized by progressive and generalized loss of skeletal muscle mass and strength3. It can lead to mobility problems, an increased risk of falls and fractures, impaired ability to perform activities of daily living, disability and loss of independence in the elderly. According to a working definition proposed by the European Working Group on Sarcopenia in Older People (EWGSOP), the criteria for diagnosis of sarcopenia is based on documentation of low muscle mass with either poor muscle strength or low physical performance4. Sarcopenia has been proposed to be primarily due to aging or secondary to reduced activity, such as bed rest, chronic diseases, malignancy, poor nutrition or endocrinal diseases5,6. The objective of the present study was to determine the prevalence of sarcopenia in the rural elderly population of South India.\n\n\nMethods\n\nThe present study was conducted in the coastal district of Dakshina Kannada in Karnataka. Karnataka is the 9th biggest state and according to the Census 2010, the geriatric population (>60 years) was 7.9%, 7th in India in terms of percentage of older persons.\n\nElderly persons >60 years of age2.\n\nA cross sectional study was conducted between July 15 and September 15, 2018.\n\nPrevalence of sarcopenia in India, according to a multi-continent study, was found to be 17.5%7. With a margin of error as 5% and 95% confidence level, the required sample size was 222. Considering a non-enrolment rate of 10%, a total of 240 participants were enrolled.\n\nParticipants were approached using a door-to-door method. All family members above 60 years were approached for enrolment. The enrolment was started from the nearest village in the field practice area and it continued until the sample size was reached in the next contiguous village.\n\nAll individuals above 60 years of age were included. Participants who were unable to walk, very frail, had cardio-pulmonary problems, which can lead to restriction of examination of muscle function, were advised for a consultation in the nearby tertiary care hospital and excluded from the study.\n\nA survey was conducted for those enrolled in the study. Variables collected were age, sex, and socio-economic class based on availability of Below Poverty Line (BPL) card. Anthropometric assessment included height and weight measurements using a portable stadiometer (SECA 213) and digital weighing machine (SECA 803). Weight was measured to the nearest of 100 gm with minimum clothing, after removing belt, shoes and any heavy extra clothing. Height was measured with accuracy to the nearest 0.1cm with participant standing without footwear with weight borne evenly on both feet and head in Frankfurt plane.\n\nMuscle function using gait speed. This was done using an 8-feet (2.4 meters) walking course with no obstructions for an additional two feet at either end. This was measured using a carpenter measuring tape. The participants were made to walk at their usual speed and time taken to cover the marked 8-feet distance was clocked using a stop-watch. A cut-off of <0.8m/s was considered as ‘slow’ performance5,9.\n\nAppendicular muscle mass (ASM). This was calculated using the Lee formula:\n\nASM = (0.244 * body weight) + (7.8 * height) + (6.6 *gender) – (0.098 * age) + (race – 3.3). For Asian people this was calculated as -1.28.\n\nSkeletal muscle index (SMI) was determined by dividing ASM with height in m2. With no reference values available from any large scale studies in India, the ASM cut-offs considered were <7.0kg/m2 in men and 5.7 in women based on previous study in Brazil9.\n\nMuscle strength. Hand grip strength represented the muscle strength of the participant. We used Jamar® Plus+ Digital Hand Dynamometer (Patterson Medical, Cedarburg, WI, United Kingdom). A cut-off of <30 kg for men and <20 kg for women was considered ‘low’ muscle strength9.\n\nParticipants with sarcopenia were classified as those with poor ASM (using Lee’s equation) among all participants with either low muscle strength or slow gait speed (Figure 1). Data were analysed using Statistical Package for the Social Sciences (SPSS) for Windows, Version 23 Chicago, SPSS Inc. Data are presented as numbers (percentage).\n\nSMI, skeletal muscle index. *Lee Equation: ASM = (0.244 * body weight) + (7.8 * height) + (6.6 *gender)–(0.098 * age)+ (race – 3.3) where for Asian race it is -1.2. Skeletal Muscle index(SMI) = ASM/(height in meter).\n\nApproval was obtained from Yenepoya University Ethics Committee - 1 before the commencement of the study (YEC-1; Protocol No: 2018/110). Written informed consent was obtained from all the study participants for voluntary participation and privacy and confidentiality of the participants were ensured.\n\n\nResults\n\nFigure 1 explains the methodology of deriving the number of participants with sarcopenia. All the participants were subjected to 8 feet walk test and a gait speed of <0.8m/sec was considered as slow. All those with normal gait speed (>0.8m/sec; 109, 45.4%) underwent hand grip dynamometry. Men with grip strength <30 kg and women with grip strength <20 kg were considered as having low grip strength. Those with normal grip strength (>30kg men, >20kg women) were considered non-sarcopenic (25, 10.4%). Those with poor grip strength were assessed for ASM using Lee’s formula, which was then converted to SMI. A value of <7.0kg/m2 in men and <5.7kg/m2 in women was considered as low SMI. The number of participants with normal gait speed, low muscle strength and low SMI were considered as having sarcopenia (12, 5%). Participants with normal gait speed, low grip strength and normal SMI were considered as non-sarcopenic (72, 30%).\n\nParticipants who had slow gait speed (131, 54.6%) also had their SMI determined (hand grip strength assessment was not used for determination of sarcopenia in the slow gait speed group). Those with slow gait speed and normal SMI were considered non-sarcopenic (109, 45.4%). Those with slow gait speed and low SMI were considered sarcopenic (22, 9.2%). Thus, overall 34 (14.2%) participants were found to have sarcopenia.\n\nTable 1 describes the socio-demographic characteristics of the 240 participants enrolled in the study. Of all those having sarcopenia, 27 (79.4%) were ≤75 years, 30 (88.2%) were women, 23 (67.6%) had BPL status, and 27 (79.4%) were married.\n\n\nDiscussion\n\nThis study enrolled 240 rural elderly participants (>60 years) and found the prevalence of sarcopenia using gait speed, hand grip strength and skeletal muscle index as 14.2%. The prevalence of sarcopenia was 3.4% in men and 24.5% in women. According to a large multi-centric study conducted in nine countries, the overall prevalence of sarcopenia was 15.2% and in India this was 17.5%7. The higher prevalence in this study was probably because the age group included in the multi-centric study was ≥65 years. There are no other studies to estimate the prevalence of sarcopenia in India, to the best of our knowledge, for further comparison. In a study in Brazil, using a similar algorithm to EWGSOP, the prevalence of sarcopenia was found to be 16.1% in men and 14.4% in women9. In another study to estimate the functional measures of sarcopenia in six low and middle-income countries, India had the highest concomitant presence of poor grip strength and reduced gait speed, 33% as compared to 12.3% in South Africa10.\n\nWith no large scale surveys done so far in India, this study provides some insight into scale of problem of sarcopenia in community dwelling elderly persons. But the study has some important limitations. Firstly, the equation used to derive the appendicular skeletal mass has not been validated for the Indian population. Secondly, we do not have normative population level data on hand-grip strength in the Indian population, therefore we used the EWGSOP recommendations. Lastly, we did not validate our findings with body-composition analysis due to logistics involved in a community-based study.\n\n\nConclusion\n\nThe prevalence of sarcopenia found in the present study was 14.2% in an elderly population (<60 years of age) and was more likely to occur in women compared to men (24.5% vs 3.4%). Larger community-based surveys are required to determine the actual burden of the problem in India.\n\n\nData availability\n\nZenodo: Sarcopenia in elderly population of Karnataka, http://doi.org/10.5281/zenodo.369193911.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nUnited Nations Department of Economic and Social Affairs Population Division: World Population Prospects: The 2017 Revision, Key Findings and Advance Tables. 2017; (accessed on 25 September 2019). Reference Source\n\nGovernment of India: Elderly in India - Profile and Programmes 2016. Ministry of Statistics and Programme Implementation. Central Statistics Office. 2016; (accessed on 20 Sept 2019). Reference Source\n\nRosenberg IH: Sarcopenia: origins and clinical relevance. J Nutr. 1997; 127(5 Suppl): 990S–1S. PubMed Abstract | Publisher Full Text\n\nCruz-Jentoft AJ, Baeyens JP, Bauer JM, et al.: Sarcopenia: European consensus on definition and diagnosis: Report of the European Working Group on Sarcopenia in Older People. Age Ageing. 2010: 39(4): 412–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSayer AA, Syddall H, Martin H, et al.: The developmental origins of sarcopenia. J Nutr Health Aging. 2008; 12(7): 427–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThompson DD: Aging and sarcopenia. J Musculoskelet Neuronal Interact. 2007; 7(4): 344–5. PubMed Abstract\n\nTyrovolas S, Koyanagi A, Olaya B, et al.: Factors associated with skeletal muscle mass, sarcopenia, and sarcopenic obesity in older adults: a multi-continent study. J Cachexia Sarcopenia Muscle. 2016; 7(3): 312–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee RC, Wang Z, Heo M, et al.: Total-body skeletal muscle mass: development and cross-validation of anthropometric prediction models. Am J Clin Nutr. 2000; 72(3): 796–803. PubMed Abstract | Publisher Full Text\n\nAlexandre Tda S, Duarte YA, Santos JL, et al.: Prevalence and associated factors of sarcopenia among elderly in Brazil: findings from the SABE study. J Nutr Health Aging. 2014; 18(3): 284–90. PubMed Abstract | Publisher Full Text\n\nBrennan-Olsen SL, Bowe SJ, Kowal P, et al.: Functional Measures of Sarcopenia: Prevalence, and Associations with Functional Disability in 10,892 Adults Aged 65 Years and Over from Six Lower- and Middle-Income Countries. Calcif Tissue Int. 2019; 105(6): 609–618. PubMed Abstract | Publisher Full Text\n\nBhargava M, Harshitha R, Shaikh N: Sarcopenia in elderly population of Karnataka. 2020. http://www.doi.org/10.5281/zenodo.3691939"
}
|
[
{
"id": "67830",
"date": "24 Jul 2020",
"name": "Min Yang",
"expertise": [
"Reviewer Expertise Sarcopenia",
"myosteatosis",
"frailty",
"geriatric syndromes",
"evidence-based medicine."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this manuscript. It addressed an old topic in the research field of sarcopenia and did not provide any valuable information for our readers. Here are my considerations.\nThis work did not cite the updated information from the current literature. For example, they cited the information from the EWGSOP that published 10 years ago. In fact, the EWGSOP2 has been published in 2018. Besides, the AWGS 2019 and other updated literature were not cited.\n\nThe method of sampling likely induced selection bias.\n\nThe diagnostic criteria of sarcopenia were not appropriate. The ASM was calculated based on anthropometric measures, which were not recommended by any consensus on sarcopenia. Because this method can not accurately estimate skeletal muscle mass. In addition, the cutoffs of the three components of sarcopenia were based on a Brail study. This is wired. Why did the authors not set these cutoffs according to the AWGS 2019 (or AWGS 2014), since the Indians are Asian? Moreover, a 2.4-meters walking test was performed to measure gait speed. The walking course is too short. Why did the authors not apply the 4-meter or 6-meter walking tests, which are the most widely used tests and are recommended by international consensuses such as EWGSOP, AWGS, FNIH, and SCWD?\n\nBecause the diagnostic criteria of sarcopenia in this study were inappropriate, the prevalence of so-called 'sarcopenia' in this study would make no sense and might mislead our readers.\n\nThis study did not report sufficient details of methods and analysis provided to allow replication by others. For example, the method to measure handgrip strength was not clearly reported.\n\nThe introduction was too short. The authors did not tell us why it is important to conduct this study.\n\nThe discussion section did not offer our readers valuable information.\n\nThere are some typos in the main text. For example, \"elderly population (<60 years of age) \" in the conclusion section (it should be >= 60 years of age). Besides, \"the ASM cut-offs considered were <7.0kg/m2 in men and 5.7 in women \" in the method section (it should be SMI instead of ASM).\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "87833",
"date": "04 Aug 2021",
"name": "Harnish P. Patel",
"expertise": [
"Reviewer Expertise Sarcopenia",
"frailty",
"comprehensive geriatric assessment"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for this manuscript. The authors are to be commended for making the effort to conduct this study. Sarcopenia is and remains a burden for many of our older population, irrespective of ethnicity so studies like these are very much needed.\nHowever, the study has many flaws that preclude it from being indexed.\nComments below:\nPlease use the term older rather than elderly.\n\nData are based on the EWGSOP (1) diagnostic algorithm published in 2010. This has been updated in 2019 (EWGSOP2) and the authors must discuss the rationale for using the older definition or present a comparative analysis. Indeed, was there a reason not to use the AWGS/AWGS(2)?\nAbstract:\nPlease present frequency and percentages.\n\nWhy was the study important?\nIntroduction:\nPerhaps too short?\n\nNeeds to mention the newer definitions of sarcopenia based on the importance of strength measures.\n\nSARC-F is recommended as initial screening.\n\nReference 3 was the original definition of sarcopenia based on mass (loss of flesh) but not strength. The definitions have been refined over time and the EWGSOP/AWGS/FNIH/IWG were instrumental in trying to reach a consensus definition (we have still not reached a global consensus definition).\n\nMention the close associations with falls and frailty.\n\nWhy is it important to understand the burden of sarcopenia in this population? What could be the driving factors of poverty - malnutrition, education, financial? What are the influencing factors on sarcopenia that would inform a public health message?\nMethods:\nStick to 'older' people rather than geriatric population. It is correct that the specialty is Geriatric Medicine but always refer to 'older people'.\n\n<0.8m/s is slower gait speed rather than low physical performance.\n\nDoes the Lee formula only measure total skeletal muscle mass? If so how did the authors arrive at ASM?\n\nHow was grip measured and what standard procedure did the authors use to avoid introducing measurement bias?\nResults:\nTable one is sparse on information - Needs values of grip strength, total skeletal muscle mass, ASM, SMI, gait speed. Were there any significant differences in these values between non sarcopenics and sarcopenics?\n\nAgain what defines poverty line?\nDiscussion:\nThe discussion is not complete.\n\nWhat was the rationale for using EWGSOP? Why was this study important? I understand that the study is unique, but how reproducible is it?\n\nIs knowing the burden of sarcopenia above poverty/malnutrition/disease a priority from LMIC counties? This is perhaps a contentious question. If the public health message is to reduce the burden of sarcopenia, should the focus be on dealing with the factors that contribute to it - malnutrition for example?\nReferences:\nThe reference list is quite short and needs to include several studies or meta-analyses that report on worldwide sarcopenia prevalence as well as prevalence in LMIC.\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?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-175
|
https://f1000research.com/articles/8-1661/v1
|
19 Sep 19
|
{
"type": "Research Article",
"title": "Ventilatory compensation during the incremental exercise test is inversely correlated with air trapping in COPD",
"authors": [
"Rottem Kuint",
"Neville Berkman",
"Samir Nusair",
"Rottem Kuint",
"Neville Berkman"
],
"abstract": "Background: Air trapping and gas exchange abnormalities are major causes of exercise limitation in chronic obstructive pulmonary disease (COPD). During incremental cardiopulmonary exercise testing, ventilatory equivalents for carbon dioxide (VE/VCO2) and oxygen (VE/VO2) may be difficult to identify in COPD patients because of limited ventilatory compensation capacity. Therefore, we aimed to detect a possible correlation between the magnitude of ventilation augmentation, as manifested by increments in ventilatory equivalents from nadir to peak effort values and air trapping, detected with static testing.\n\nMethods: In this observational study, we studied data obtained previously from 20 COPD patients who, during routine follow-up, underwent a symptom-limited incremental exercise test and in whom a plethysmography was obtained concurrently. Air trapping at rest was assessed by measurement of the residual volume (RV) to total lung capacity (TLC) ratio (RV/TLC). Gas exchange data collected during the symptom-limited incremental cardiopulmonary exercise test allowed determination of the nadir and peak effort values of VE/VCO2 and VE/VO2, thus enabling calculation of the difference between peak effort value and nadir values of VE/VCO2 and VE/VO2, designated ΔVE/VCO2 and ΔVE/VO2, respectively. Results: We found a statistically significant inverse correlation between both ΔVE/VCO2 (r = -0. 5058, 95% CI -0.7750 to -0.08149, p = 0.0234) and ΔVE/VO2 (r = -0.5588, 95% CI -0.8029 to -0.1545, p = 0.0104) and the degree of air trapping (RV/TLC). There was no correlation between\n\nΔVE/VCO2 and peak oxygen consumption, forced expiratory volume in the first second, or body mass index. Conclusions: The ventilatory equivalents increment to compensate for acidosis during incremental exercise testing was inversely correlated with air trapping (RV/TLC) and may be a candidate prognostic biomarker.",
"keywords": [
"air trapping",
"COPD",
"exercise",
"isocapnic buffering phase",
"ventilatory equivalent"
],
"content": "Abbreviations\n\nBMI, body mass index; COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in the first second; GOLD, global initiative for chronic obstructive lung disease; IC, inspiratory capacity; RV, residual volume; TLC, total lung capacity; VCO2, CO2 output; VE, minute ventilation; VE/VCO2, ventilatory equivalent for CO2; VE/VO2, ventilatory equivalent for oxygen; ΔVE/VCO2, nadir to peak value of VE/VCO2; ΔVE/VO2, nadir to peak value of VE/VO2; VO2, oxygen consumption; VT, ventilatory threshold.\n\n\nIntroduction\n\nChronic obstructive pulmonary disease (COPD) patients often demonstrate significant effort limitation, chiefly resulting from gas exchange abnormalities and ventilation-perfusion mismatching1. This situation is often compounded by hyperinflation and air trapping with dynamic hyperinflation during exercise and gradual reduction of inspiratory capacity2. To evaluate exercise capacity and determine the degree of effort limitation and its mechanisms, the incremental exercise test is often applied, during which several ventilatory events occur and draw close attention.\n\nDuring incremental cardiopulmonary exercise testing, the ratio of minute ventilation (VE) to carbon dioxide output (VCO2) and to oxygen consumption (VO2), also known as ventilatory equivalent for carbon dioxide (VE/VCO2) and oxygen (VE/VO2), respectively, serve to evaluate ventilatory efficiency. The ventilatory threshold (VT), a term coined in the context of gas exchange measurements and represents to a great extent the VO2 at anaerobic threshold, heralds the onset of the isocapnic buffering phase, in which there is an increased contribution of anaerobic metabolism to provide the energy required for the increasing demands of exercise, accompanied by increased CO2 production with corresponding ventilation increase. With the accumulation of lactic acid and H+ protons, as the bicarbonate reserves are exhausted beyond the isocapnic buffering phase, an augmented ventilatory response starts at the ventilatory compensation point, which is disproportionate to the degree of CO2 production, leading to VE/VCO2 surge towards peak effort, a phenomenon termed the ventilatory compensation phase.\n\nThe VE/VCO2 is often increased as a result of ventilation-perfusion mismatching1,3. The nadir VE/VCO2 value during incremental exercise is inversely related to the degree of effort limitation4, as well as the severity of airway obstruction, as determined by the values of forced expiratory volume in first second (FEV1)5. The nadir value is considered to reflect ventilatory efficiency as higher values obtained at the early stages of the incremental exercise test result from frequently encountered hyperventilation. Interestingly, marked hyperinflation and reduced ventilatory capacity tend to reduce ventilatory equivalents as ventilation may become constrained. Both baseline and peak effort VE/VCO2 values were found to be lower in patients with more severe airway obstruction as VE is decreased relative to VCO2 in these patients6.\n\nAlthough COPD patients may terminate exercising at an early stage during the incremental exercise test due to airflow obstruction and air trapping, suboptimal effort and deconditioning, the latter of which may be associated with somewhat earlier development of metabolic acidosis, may also lead to early termination of the exercise test. Regardless of the reason causing decreased exercise performance, the ventilatory compensation phase may not be demonstrated. Ventilatory equivalents during incremental exercise may be affected by the above-mentioned processes in different directions. In this study we sought to confirm the presumption that the ability to achieve ventilatory compensation in response to acidosis is related to the degree of air trapping, with the hypothesis that the ability to augment ventilation beyond the ventilatory compensation point in COPD patients is inversely correlated with the degree of air trapping.\n\n\nMethods\n\nA retrospective analysis of data obtained from the medical records of COPD patients who underwent incremental cardiopulmonary exercise testing in the pulmonary function laboratory of the Institute of Pulmonary Medicine in the Hadassah Medical Center, Jerusalem, Israel between June 2010 and August 2016, and in whom whole body plethysmography was performed concurrently, as part of their routine clinical evaluation. The number of patients was determined by the availability of incremental exercise test and plethysmography data that was performed concurrently. Subjects were previously diagnosed with COPD based on post-bronchodilator spirometry showing an FEV1 to forced vital capacity (FVC) ratio (FEV1/FVC) of ≤0.7, regardless of the value of FEV11 (according to 2017 GOLD guidelines). Comorbidities such as obesity, cardiac disease and use of beta blockers were noted, but were not a cause to exclude patients from the study. The research ethics board (REB) of the Hadassah Medical Organization (protocol No. 0040-16-HMO) approved data collection and waived the need for informed consent, as this research was retrospective, did not affect patient management, or involve collecting biospecimens.\n\nPulmonary function tests and exercise tests were performed by technicians in the presence of a physician during exercise tests. Whole-body plethysmography was performed using a commercially available body plethysmograph (Elite series, MedGraphics). Spirometry performance and slow vital capacity determination were followed by assessment of lung volumes, which was performed by direct measurement of thoracic gas volume (TGV), from which the residual volume (RV), total lung capacity (TLC) and RV/TLC ratios could be calculated as the primary method to estimate gas trapping. Additionally, we determined the ratio of inspiratory capacity (IC) to total lung capacity, IC/TLC, which has been proposed to reflect lung expansion as a result of reduced lung recoil in emphysema7. Using the same lung function system, transfer factor of the lung for carbon monoxide (TLCO) was measured by the single breath method.\n\nPatients performed incremental symptom-limited cycle ergometry connected to a metabolic system with cycle ergometer (Ultima CardiO2, MedGraphics). For each patient, baseline measurements were obtained during the resting period of 1 minute, after which the patient would start cycling at a constant rate of 50–60 rpm. Following a 1–2 minute warm-up period at 0 watts and with VO2 and VCO2 reaching a plateau, a ramp protocol was started with a workload increase of 15–25 watt per minute, depending on the predicted maximal VO2 and the general state of the patient. The test was terminated at exhaustion. Routine precordial 12-lead electrocardiogram monitoring, continuous measurements of VE, VO2, VCO2 (averaged every 10 seconds), heart rate and finger arterial pulse oximetry were recorded. The peak VO2 and the VT were both noted, as well as nadir ventilatory equivalents (VE/VCO2 and VE/VO2). Values of VE/VCO2 and VE/VO2 obtained at the termination of loaded exercise (peak effort) enabled calculation of the difference between peak effort value and nadir values of VE/VCO2 and VE/VO2 (designated ΔVE/VCO2 and ΔVE/VO2, respectively).\n\nStatistical analysis was performed using the GraphPad Prism 3.0 program software and Spearman’s test was used for correlation analysis, with calculation of the Pearson correlation coefficient (r) between measured parameters. Correlations were considered of statistical importance if two-tailed p value was <0.05. Unpaired t-test was used to compare between groups, and a two-tailed p value < 0.05 was considered statistically significant\n\nThis manuscript is compliant with the STROBE guideline.\n\n\nResults\n\nIn total, 20 patients were included in our analysis with a mean ± SD age of 63 ± 10 years (range 37–77). None of the patients had cardiac failure. Two patients were receiving beta blockers. Table 1 summarizes clinical data of these patients.\n\nValues are mean ± SD.\n\nBMI, body mass index; FEV1, forced expiratory volume; FVC, forced vital capacity; HR, heart rate; GOLD, Global initiative for chronic Obstructive Lung Disease; IC, inspiratory capacity; RV, residual volume; TLC, total lung capacity; TLCO, transfer factor of the lung for carbon monoxide; VO2, oxygen consumption; VE/VO2, ventilatory equivalent for oxygen; VE/VCO2, ventilatory equivalent for carbon dioxide; Δ is the difference between measured value of ventilatory equivalent at peak effort and nadir value.\n\n*Breathing reserve = (maximal voluntary ventilation - peak ventilation) / maximal voluntary ventilation.\n\nWe found a statistically significant inverse correlation between both ΔVE/VCO2 (r = -0.5058, 95% CI -0.7750 to -0.08149, p = 0.0234) and ΔVE/VO2 (r = -0.5588, 95% CI -0.8029 to -0.1545, p = 0.0104), with the degree of air trapping as estimated by the RV/TLC ratio (Figure 1). On the contrary, there was no correlation between the nadir values of the ventilatory equivalents and RV/TLC (Table 2). Interestingly, inspiratory capacity to total lung capacity ratio (IC/TLC), which reflects pulmonary expansion in emphysema, did not show a correlation with either ΔVE/VCO2 or ΔVE/VO2 (Table 2).\n\nCorrelation between ΔVE/VCO2 (A), ΔVE/VO2 (B) and RV/TLC. RV/TLC, residual volume to total lung capacity ratio; ΔVE/VCO2, nadir to peak increment of ventilatory equivalent for CO2; ΔVE/VO2, nadir to peak increment of ventilatory equivalent for oxygen. p<0.05 points to a statistically significant correlation.\n\np value <0.05 is considered of statistical importance.\n\nr, Pearson correlation coefficient; BMI, body mass index; IC, inspiratory capacity; RV, residual volume; TLC, total lung capacity; FEV1, forced expiratory volume; TLCO, transfer factor of the lung for carbon monoxide; VO2, oxygen consumption; VE/VCO2, ventilatory equivalent for carbon dioxide; VE/VO2, ventilatory equivalent for oxygen; Δ is the difference between measured value at peak effort and nadir value\n\nWe examined possible correlation between ΔVE/VCO2 and other static parameters of pulmonary function or findings on exercise testing (summarized in Table 2). Notably, we found no significant correlation between neither ΔVE/VCO2 nor nadir VE/VCO2 and peak VO2 or FEV1. As expected1, nadir values of VE/VCO2 correlated with TLCO (r = -0.5281, 95% CI -0.7923 to -0.09708, p = 0.0201); however, we did not find a correlation between ΔVE/VCO2 and TLCO (Table 2).\n\nIn an attempt to identify reasons for the lack of increment of ΔVE/VCO2 besides factors related to air trapping, we compared values of parameters related to static pulmonary functions and those related to performance of exercise in patients with and without increment in VE/VCO2 (i.e., ΔVE/VCO2 = 0) (Table 3). While FEV1, IC/TLC, TLCO, peak VO2, and nadir VE/VCO2 were not different between these two groups, RV/TLC and peak respiratory exchange ratio had a statistically important difference between the two groups. However, these latter two factors are inherently derived from the parameters that the correlation study had examined and therefore, this difference is not surprising.\n\nValues are mean ± SD. p<0.05 signifies an important difference.\n\nRER, respiratory exchange ratio; RV, residual volume; TLC, total lung capacity; TLCO, transfer factor of the lung for carbon monoxide; VO2, oxygen consumption; FEV1, forced expiratory volume; IC, inspiratory capacity; VE/VCO2, ventilatory equivalent for CO2; ΔVE/VCO2 is the difference between measured value at peak effort and nadir value of VE/VCO2.\n\n\nDiscussion\n\nInefficient ventilation in patients with COPD and particularly emphysema is often evidenced by elevated nadir VE/VCO2. The elevation of nadir VE/VCO2 may be related to the extent and severity of emphysematous changes in the lungs. It has been shown that in patients with mild to moderate airflow decrease, the values of nadir VE/VCO2 correlated with the percentage of low attenuation areas on computerized tomography8,9 as well as the decrease in pulmonary perfusion, as estimated by the inert gas rebreathing method9. Interestingly, in smokers without COPD, the degree of CO diffusion decrease correlated with elevation of nadir VE/VCO2 during the incremental exercise test10, again pointing to a strong relation between impaired ventilation efficiency and gas exchange ability. Notably, significantly increased ventilatory constraints, as occurs in marked air trapping, decrease VE, thus leading to lower nadir VE/VCO2 in more severe airway obstruction2,6.\n\nIn this study we examined the ability of COPD patients during incremental exercise testing to increase ventilation towards peak effort, focusing on the increase in the VE/VCO2 and VE/VO2 slopes that occur following the ventilatory compensation point. We quantified this phenomenon measuring the difference between the slope values of VE/VCO2 and VE/VO2 at nadir and peak effort obtained post the ventilatory compensation point, which we termed ΔVE/VCO2 and ΔVE/VO2, respectively. We found that the ability to increase ventilation during incremental exercise testing in response to metabolic acidosis, as normally occurs in higher exercise intensities beyond the ventilatory compensation point, was diminished in COPD patients and in correlation with the severity of air trapping as represented by RV/TLC obtained at rest. Therefore, inability to compensate for the increased ventilatory demands of acidosis may contribute to termination of effort at lower exercise intensity. Furthermore, this may implicate a difficulty in coping with the ventilatory demands of metabolic acidosis resulting from states such as acute renal failure or septic shock, thus ΔVE/VCO2 (and seemingly ΔVE/VO2) may allow assessment of the ability of COPD patients to withstand such metabolic challenges.\n\nOne way in which pulmonary hyperinflation can be expressed is by the increased ratio of RV/TLC, which reflects air trapping and is associated with airway narrowing. Alternatively, IC/TLC may also be decreased because of lung expansion associated with emphysematous changes and reduced lung recoil. A recent study showed that either increased RV/TLC or decreased IC/TLC or both simultaneously can be present, with airway narrowing on imaging being associated with increased RV/TLC and emphysema associated with deceased IC/TLC7. Therefore, it seems that the lack of increments in ventilatory equivalents in our study can be related to air trapping but not to the mere presence of emphysema.\n\nOne may argue that the limited rise of ventilatory equivalents beyond the ventilatory threshold during the incremental exercise test may reflect sub-optimal exercise performance; however, we think that the lower respiratory exchange ratio at peak exercise represents another manifestation of the decreased ventilatory response.\n\nAlthough both ΔVE/VO2 and ΔVE/VCO2 seem to show similar correlation to RV/TLC and utility in estimating the ventilation response to acidosis under these conditions, a further study with larger numbers of patients may help distinguish the different roles of these parameters.\n\nThis study has several limitations. The retrospective design and the small number of patients included are important limitations, in particular when comparing two subgroups. However, these limitations may be of lesser significance when searching for correlations. Some patients were obese, which may have caused mild restriction, somewhat contributing to air trapping. However, we didn’t find a correlation between body mass index (BMI) and either ΔVE/VCO2 or ΔVE/VO2. The group with the greater ventilatory equivalents increment had a nearly significant higher nadir VE/VCO2 value (p = 0.53), suggesting a possible baseline difference between those who develop an increment versus those who don’t. This could be explained along the same line in which it is suggested that those patients who can develop an augmented ventilatory response, i.e. increase VE, are also able to produce a ventilatory equivalent increment towards peak effort. Unfortunately, dynamic hyperinflation, a well-known phenomenon that is associated with ventilatory constraints during exercise in COPD, was not assessed and therefore, we cannot comment on the relation between dynamic hyperinflation (i.e. changes in IC/TLC) and ventilatory equivalents increment in this cohort.\n\nIn conclusion, ventilation augmentation during incremental exercise testing at exercise intensities beyond the ventilatory compensation point in COPD patients was diminished in correlation with the severity of air trapping. Future studies will confirm the clinical usefulness of measuring ΔVE/VCO2 and ΔVE/VO2, including their potential role as serially measured physiologic parameters to assess effects of interventions (e.g. pulmonary rehabilitation) in COPD.\n\n\nData availability\n\nHarvard Dataverse: Ventilatory Equivalents increment during Exercie in COPD. https://doi.org/10.7910/DVN/QTCV0M11.\n\nThis project contains the following underlying data:\n\n- nadir to peak VE-VCO2 during exercise in COPD.tab (demographic information and parameter measurements for each patient).\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\nExercise tests and static pulmonary function tests were performed by medical technicians Ahuva Mizrachi, Ruhama Erental, Elat Bardach and supervised by Samir Nusair.\n\nNote: This work has been presented in part, in poster form, during the European Respiratory Society International Congress 2016, London, United Kingdom.\n\n\nReferences\n\nWasserman K: Diagnosing cardiovascular and lung pathophysiology from exercise gas exchange. Chest. 1997; 112(4): 1091–1101. PubMed Abstract | Publisher Full Text\n\nO'Donnell DE, Revill SM, Webb KA: Dynamic hyperinflation and exercise intolerance in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2001; 164(5): 770–777. PubMed Abstract | Publisher Full Text\n\nPaoletti P, De Filippis F, Fraioli F, et al.: Cardiopulmonary exercise testing (CPET) in pulmonary emphysema. Respir Physiol Neurobiol. 2011; 179(2–3): 167–173. PubMed Abstract | Publisher Full Text\n\nCaviedes IR, Delgado I, Soto R: Ventilatory inefficiency as a limiting factor for exercise in patients with COPD. Respir Care. 2012; 57(4): 583–589. PubMed Abstract | Publisher Full Text\n\nNeder JA, Arbex FF, Alencar MC, et al.: Exercise ventilatory inefficiency in mild to end-stage COPD. Eur Respir J. 2015; 45(2): 377–387. PubMed Abstract | Publisher Full Text\n\nThirapatarapong W, Armstrong HF, Thomashow BM, et al.: Differences in gas exchange between severities of chronic obstructive pulmonary disease. Respir Physiol Neurobiol. 2013; 186(1): 81–86. PubMed Abstract | Publisher Full Text\n\nSmith BM, Hoffman EA, Basner RC, et al.: Not all measures of hyperinflation are created equal: lung structure and clinical correlates of gas trapping and hyperexpansion in COPD: the Multi-Ethnic Study of Atherosclerosis (MESA) COPD Study. Chest. 2014; 145(6): 1305–1315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCrisafulli E, Alfieri V, Silva M, et al.: Relationships between emphysema and airways metrics at High-Resolution Computed Tomography (HRCT) and ventilatory response to exercise in mild to moderate COPD patients. Respir Med. 2016; 117: 207–214. PubMed Abstract | Publisher Full Text\n\nJones JH, Zelt JT, Hirai DM, et al.: Emphysema on Thoracic CT and Exercise Ventilatory Inefficiency in Mild-to-Moderate COPD. COPD. 2016; 14(2): 210–218. PubMed Abstract | Publisher Full Text\n\nWalter Barbosa G, Neder JA, Utida K, et al.: Impaired exercise ventilatory efficiency in smokers with low transfer factor but normal spirometry. Eur Respir J. 2017; 49(3): pii: 1602511. PubMed Abstract | Publisher Full Text\n\nKuint R, Berkman N, Nusair S: \"Ventilatory Equivalents increment during Exercie in COPD\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/QTCV0M"
}
|
[
{
"id": "56449",
"date": "02 Dec 2019",
"name": "Gabriele Valli",
"expertise": [
"Reviewer Expertise Exercise Physiology - Hypoxia - Ventilatory Response to Exercise and High Altitude - Critical Care and Emergency Medicine"
],
"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 present study, the authors investigate a possible correlation between the magnitude of ventilation augmentation (manifested by increments in ventilatory equivalents from nadir to peak effort) and the lung air trapping indexes measured at rest. The authors analyzed retrospectively data from 20 symptom-limited incremental exercise test performed by 20 COPD patients. Patients underwent to CPET as part of a routine evaluation. The differences between nadir to peak exercise of VE/CO2 were correlated to air trapping parameters measured at rest in a plethysmography. A positive correlation was found between nadir to peak exercise VE/CO2 and VE/VO2 and the degree of air trapping expressed as RV/TLC. The authors concluded that ventilatory equivalents increment (above anaerobic threshold) could be inversely correlated with air trapping and it may be a candidate prognostic biomarker.\n\nAlthough the paper is well written I have some comments to do:\nThe authors choose to calculate ventilatory equivalent subtracting the values at peak exercise to the values measured at the nadir, assuming that the reduced increment of ventilatory equivalent after the minimum values could be mainly referred to a reduced ventilatory capacity of the subject due to air trapping measured at rest. That could be right but it is not completely correct. The simple correlation measured by the authors does not completely clarify the reasons for the interruption of the test by patients, above or below the respiratory compensation point (RCP). Some studies had demonstrated a reduced ventilatory compensation capacity in patients with a more severe emphysema i.e Paoletti et al, 20111 nerveless it should take in account that several metabolic alterations occur after the anaerobic threshold that are independent to the ventilatory mechanics and that are more closely related to physical conditioning of the patient. After the anaerobic threshold, peak ventilatory response could be related to several different causes: the exercise intensity and the power increments, the anaerobic reserve of the subject, the muscular O2 extraction capacity, the amount of metabolic acidosis, the increase in catecholamine levels, the developed or not of dynamic hyperinflation, the ventilatory chemiosensitivity of the carotid bodies, some cardiovascular alteration as an opening of PFO or the develop of pulmonary hypertension, even the individual motivation to complete the exercise and the subject propensity to perform that specific exercise. It is difficult to understand which is the real cause of exercise interruption and this could significantly affect the amount of nadir to peak ventilatory equivalent difference. The difference between nadir to peak ventilatory equivalents does not seems to be a good estimate of patient’s ventilatory compensation during the exercise.\n\nIt should be noted that, in the regression analysis there are half of the patients that did not increase ventilatory equivalents, which means that they stop the exercise at RCP or immediately after. Table 3 show a significant difference in peak RER between the patients who have not shown increments in VE/CO2 and the ones who have continued to increase ventilation, suggesting that the two groups reach different intensity exercise domain and probably are different populations (i.e. RC/TLC significantly different). A more complete table, that includes Watt Max, VO2 peak in ml/kg, VO2 pulse, VE/MVVpeak and FEV1/FVC of the two groups could be helpful. Unfortunately it is impossible to know why the subject who showed no increase in VE/VCO2, interrupt the exercise immediately after the reach of RCP. To put all the results in the same regression appear as an hazard, there are too many unpredictable sources of error. The authors must provide consistent evidence that the observed correlation is not only apparent. The estimated coefficients of the linear regression and the relative interval of confidence should be calculated. An estimation of the standard error of regression coefficient R2 should be provide together with the 95% confidence band for R. A residual analysis could help to understand how good is the correlation and if the assumption of normality of the distribution is preserved.\n\nThe variable chosen by the authors to represent the exercise ventilatory compensation of the patients is not the one universally accepted and utilized for this propose. The authors should clarify why they have not utilized, for instance, the VE/CO2 slope. In a recent paper Gargiulo P. and coworkers demonstrated that the analysis of VE/CO2 slope and its intercept on the abscissa (VEint), that represent the ventilatory requirements when pulmonary gas exchange is null, represents better the ventilatory efficiency (Gargiulo P et al. 20142). In COPD patients VEint correlate with the VD and Neder and colleagues recently showed that increases in VEint better reflected the progression of functional impairment form mild to end-stage of COPD (Neder JA et al. 20153). I suggest to test also these parameters in you analysis that are more clearly related to pulmonary efficiency and that are less dependent from the individual motivation of the patients to tolerate the effort.\n\nThe authors conclude the abstract suggesting that the reduced ventilatory increment in VE/VCO2 from rest to peak exercise could be a prognostic biomarker. This assumption is not clarified in the text, no data was provided to support this statement and the assumption remains speculative.\n\nMinor comments:\n\nAbstract\n\nLine 4, p 1_ “During incremental cardiopulmonary exercise testing, ventilatory equivalents for carbon dioxide….. may be difficult to identify in COPD patients because of limited ventilatory compensation capacity”. This is not correct. Ventilatory equivalents are always identifiable, in COPD patients could be difficult to identify the respiratory compensation point, the point at which it could be observed an hyperventilatory response to late-exercise acidosis.\n\nLine 19, p 1_ “DVE/VCO2 and DVE/VO2”. DVE/VCO2 abbreviation is usually utilized to indicate VE/CO2 slope and could be confounding, I suggest to change it with a new acronym that specifies that it is the difference between the peak and nadir values.\n\nLine 28, p 1_ “…and may be candidate prognostic biomarker” the statement is speculative and not investigated in the study.\n\nIntroduction\n\nLine 26, p 3 _ “The ventilatory threshold (VT)…. “. I suggest to utilize a different abbreviation (i.e. AT or LT), “VT” could be confused with Tidal Volume.\n\nMethods Lines 1-10, p 4 _ “Patients performed incremental symptom-limited cycle ergometer…” The authors mentioned a 1-2 minute of warm-up period at 0 watt. 0 watts assumes that an ergometer with pedal assistance has been used. What kind of cycle ergometer was used? did you mean a period of free pedaling at the minimum of watts? Have you estimated the amount of the minimum load of your cycle-ergometer? 1-2 min of warm up could be correct with a real 0 watts ergometer but it could be a too short period if the minimum load provide by the ergometer was 20W or greater.\n\nLine 10, p4_ “ the test was terminated at exhaustion” Can you please better define “exhaustion”?\n\nResults\n\nTable 1. The table should be integrated with the absolute value of VO2 peak in ml/kg, VO2@AT in ml/kg, Watt Max and RER at peak exercise. I suggest to put SpO2 peak rather than the desaturation at peak effort. An explication of how the authors calculated BR should be added to Methods. The percentage of tests in which the respiratory compensation point was identifiable should be added somewhere. DVE/CO2 should be change with some other abbreviation.\n\nLines 5-16, p4 _ “ DVE/VCO2 (r= -0.5058, 95% CI -0,7750 to – 0.08149, p =0.0234)…” a p = 0.0234 looks quite significantly, nerveless the adaptation of the regression appear very low, it seems that R2 is not more than 0.25. I suggest to add to your plot the regression coefficients with the respective standard errors. It could be useful analyzed the residual plot in order to increase the adaptation.\n\nLines 19-20, p4. “Notably, we found no significant correlation between neither DVE/VCO2 nor nadir VE/VCO2 and peak VO2 or FEV1. \". Do you mean the absolute values or % predicted?\n\nTable 3. The table shows a significant difference in RER peak between the two population analyzed. The patients with a significant increase in DVE/VCO2 probably achieve a greater effort intensity. As the entire correlation depends on the patients who sustain the effort beyond their VEVCO2 nadir , it is likely that the correlation is no longer detectable in the more severe patients. I would like to know the differences between the two groups in VO2 peak expressed as ml/kg, Watt Max, BR%, Heart Rate Reserve (220-age), VO2 pulse at peak exercise, FEV1 in mL and FEV1/VC.\n\nDiscussion\n\nLines 17-19, p 5. “In this study we examined the ability of …. Focusing on the increase in the VE/VCO2 and VE/VO2”. This is not correct. You have not measured any slope. You measure just a difference.\n\nLines 14-19. P 6. “Therefore the inability to compensate…”. This statement is entirely speculative, beyond the scope of the study and not supported by any data that can be highlighted in the paper.\n\nLines 28-34, p 6. “Therefore, it seems that the lack of increment….” You are comparing a static measurement with a dynamic alteration that occurs in the moment of maximum stress. The problem is that you have not measured any indexes of dynamic hyperinflation. Your conclusion cannot be proven.\n\nLines 34-35, p. 6 “however, we think that the lower respiratory exchange ratio at peak exercise represents another manifestation of the decreased ventilatory response”. Unfortunately it does not stand out easily if the patient has stopped due to actual exhaustion or to other reasons. Especially in a retrospective study.\n\nLines 49-50, p. 6 “Some patients were obese, which may have caused mild restriction…” You should take in account that BMI could largely affect the VCO2 body storage and, as a consequence, the RCP detection or VE/VCO2 peak values. Is there any differences between the two groups (No Nadir to Peak increment vs patients with nadir to peak increment) in BMI? Can you provide this data?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "5277",
"date": "10 Mar 2020",
"name": "Samir Nusair",
"role": "Author Response",
"response": "The authors would like to thank Dr. Gabriele Valli for his valuable comments. This is a retrospective study with a small number of patients, and we certainly acknowledge the inherent significant limitations, which we refer to in the Discussion section. We sought to study changes in VE/VCO2 during the incremental exercise test in COPD patients, a very intriguing subject. Furthermore, we consider this work as a preliminary/exploratory study, and as that it has well acknowledged limitations and implications. Furthermore, we added data of VE/VCO2 slope and VE/VCO2 line intercept as suggested, providing further insight into our cohort. We would like to address the concerns raised by the Reviewer. Re Major Comments: 1. We certainly agree with the reviewer that there are several possible mechanisms and factors that may contribute to exercise limitation in patients with COPD. In this study we aimed at studying the changes in VE/VCO2 during exercise in these patients. We investigated a possible relationship between airway trapping (defined by increased RV/TLC) and the decreased ability to increase ventilation in response to acidosis as seen by the lack of VE/VCO2 rise and absence of the ventilatory compensation phase seen in people without COPD. We certainly do not claim cause and effect on the basis of our findings. 2. We have now added information as requested by this reviewer to Table 1 and Table 3 of peak Work Rate, peak VO2 (ml/kg/min),FEV1/FVC and exercise ventilatory mechanical data (VE/MVV, peak exercise Vt/VC). Patients who had an increment in VE/VCO2 had higher VO2 (per body weight), higher peak WR and higher RER. Again, this is not about cause and effect but epiphenomena. Whether these patients were “more motivated” to perform exercise than those who did not cannot be absolutely excluded, yet with all patients motivated to perform exercise until exhaustion, (i.e., could not keep required cycling pace) or had marked symptoms such as shortness of breath, simple lack of motivation to exercise is unlikely sole explanation. Adding data regarding end-tidal PCO2 at baseline and at peak exercise in both groups should be also helpful. We sought to find a correlation between increments of ventilatory equivalents and air trapping (see Results section, paragraph 2). We did not seek to find a linear regression. We agree with the reviewer about the “hazards” in creating a linear regression of all the data, especially when many other factors could be involved. Therefore we take a step back and present the data of correlation we intended to show originally, adding it to Table 2, in the first two rows. We deleted Figure 1 as it is irrelevant. 3. In this work, we originally wanted to look at the changes in VE/VCO2 during exercise, the isocapnic buffering phase and the ventilatory compensation phase. Previous observations have noted the absence of significant ventilatory compensation in patients with COPD (Ref 6). Since there are previous descriptions of correlation between VE/VCO2 slope and air trapping (Refs 1,3 &5 in the manuscript) we attempted to somehow quantitate the increment in VE/VCO2 during incremental exercise, expecting that some patients may not have an increment (deltaVE/VCO2=0). To the best of our knowledge this is a somewhat novel approach. In our opinion, nadir VE/VCO2 may be easier to identify in routine clinical practice and also to relate to peak VE/VCO2. In this cohort, we found a high correlation between Nadir VE/VCO2 and VE/VCO2 slope. Data of VE/VCO2 slope was added. There was a significant correlation between Nadir VE/VCO2 and VE/VCO2 slope (R=0.82 95%CI 0.59-0.92, p<0.0001).Data of VE/VCO2 intercept was also added. (All 3 Tables). In Table 3 we show that VE/VCO2 intercept was significantly higher in patients in whom deltaVE/VCO2=0. We now show that our findings are in agreement with this parameter which represents dead space breathing. 4. Reference to a “possible prognostic biomarker” in the end of the abstract was deleted. Re Minor Comments: Abstract: 1. Sentence was corrected “During incremental cardiopulmonary exercise testing, actual nadir values of ventilatory equivalents for carbon dioxide (V E/VCO 2) and oxygen (V E/VO 2) may be difficult to identify….” 2. The authors have checked before writing this manuscript the use of the symbol delta, and we did not find a similar use of it in this context, even in reference to VE/VCO2 slope, therefore we resort to continue using this symbol in our manuscript. Another acronym/abbreviation will be more difficult to use at this stage. 3. The reference to “biomoarker” was omitted. Introduction: 1. The abbreviation “VT” in relation to ventilatory threshold was deemed unnecessary at this stage and it is no longer used. Methods: 1. Sentence was re-written “Following a 1–2 minute warm-up period of unloaded exercise and with VO 2 and VCO 2 reaching a plateau,….”. We accept the reviewer’s note that this cannot be defined as WR=0, since it was pedaling against the inherent resistance of the cycle machinery without motored assistance. 2. Exhaustion in the context of exercise test performance is now defined. The sentence was re-written:“The test was terminated at exhaustion, when the patient could not keep the required cycling pace or asked for exercise termination.” Results: 1. More information added to Table 1 and other tables. Breathing reserve is defined at the bottom of Table 1.The percentage of patients with identified respiratory compensation point would be 50% and is represented by a deltaVE/VCO2>0. 2. As noted above we performed correlation estimations and not linear regression (explained above and changes described) 3. There was no correlation between neither deltaVE/VCO2 nor nadir VE/VCO2 and peak VO2 and FEV1, as percentage of predicted. We added also a correlation with VO2 in ml/kg/min, which just achieved a significant correlation with a p=0.0462. 4. Table 3 is now expanded and more parameters are compared. Discussion: 1. The sentence now has been changed “…..focusing on the increase in V E/VCO 2 and V E/VO 2 that occur following the ventilatory compensation point.” The word “slopes” was deleted. 2. The entire sentence “Therefore, inability to compensate for the increased ventilatory demands of acidosis may contribute to termination of exercise at lower exercise intensity.” was deleted. 3. The sentence has been changed and “can be related to” is changed to “is correlated with”. 4. Instead of “represents” the phrase is changed to “may represent”, thus expressing some reservation about this conclusion. 5. Comparison of BMI between the two groups has been added. There was no difference in BMI between the two groups."
}
]
},
{
"id": "57517",
"date": "09 Dec 2019",
"name": "William W. Stringer",
"expertise": [
"Reviewer Expertise Exercise Physiology",
"COPD",
"Pulmonary Function Testing"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\n\nDrs Kuint, Berkman, and Nusair retrospectively analyzed 20 COPD patients (GOLD 1: #7, II: #8, III: #4, IV: #1) who had full PFTs with lung volumes and a CPET test over a 7 year period (2010 to 2016). They were interested in air trapping and the effect on exercise performance, specifically trying to better understand the changes in Ventilatory Equivalents for Oxygen and Carbon Dioxide during incremental exercise tests on a cycle ergometer (nadir value to peak value). Further, they relate these changes to resting pulmonary function testing. The study was retrospective. They conclude that “ventilatory equivalents increment to compensate for acidosis during incremental exercise testing was inversely correlated with air trapping (RV/TLC) and may be a candidate prognostic biomarker.” The study is well written.\n\nMajor Issues\nThe study would have been greatly assisted by obtaining inspiratory capacity (IC maneuvers) during exercise to document dynamic hyperinflation.\n\nThe study is retrospective, is a very small group, and was collected across 7 years (2010-2016, about 2-3 subjects per year), meaning that there is great potential for changes in devices, calibration, technician training, etc. that may have affected the PFT and CPET measurements.\n\nThe GOLD III and IV subjects are very poorly represented (Gold III (20%) and Gold IV (5%)), i.e. most subjects (75% were Gold I or II). Per the Neder and Thirapatarapong references in your paper, Gold I-II have lower ETCO2 and higher VE/VCO2 reflecting less efficient ventilation, and GOLD III-IV have higher ETCO2 and lower VE/VCO2 reflecting mechanical limitation.\n\nThis paper is also hampered by so many of the changes in VE/VCO2 (10) and VE/VO2 (7) from peak-nadir were nil, i.e. = \"0\"\n\nThere is no analysis of VE/VCO2 slope or intercept reported in the paper or data spreadsheet.\n\nNo blood gases (to document acidosis or lactate levels) or end tidal CO2 values to document changes in ventilatory efficiency. Importantly, there is no discussion of the determinants of VE/VCO2, i.e. VE/VCO2 i.e., = k /(PaCO2) * ( 1-VD/VT) and how these two variables interact in the current study.\n\nCOPD patients are less likely to protect their arterial pH and arterial CO2 during exercise (Casaburi, et al, Am Rev Respir Disease, 143: 9-18, 19911) See figure # 8. There is no discussion of this important feature of COPD patients in the manuscript.\n\nMinor Issues\nIntroduction, page 3, First Paragraph, line 2. “Effort” should likely be exercise or activity.\n\nIntroduction, page 3, First Paragraph, line 3+4. Here you mention gas exchange abnormalities and ventilation perfusion mismatching, but in paragraph 4, lines 3 you also discuss deconditioning. All three mechanisms appear to be important and should be mentioned in both places.\n\nIntroduction, page 3, Second Paragraph, line 14. “exhausted” should be “decreasing”.\n\nIntroduction, page 3, Third Paragraph, line 6 and 14. Reference 5 and 6 would both seem to contribute to the concepts here, and should both be quoted.\n\nMethods, Paragraph 1, Study Design and Subjects, lines 1-5. How are the authors confident that all subjects with COPD and full PFTs studied in the laboratory were identified for the current manuscript?\n\nMethods, Paragraph 1+2, Procedures. Were there any changes in PFT or CPET equipment, calibration, or technician protocols during the 7 years of data collection that might affect spirometry, lung volumes, gas transfer or CPET variables?\n\nMethods, Paragraph 2, Procedures. 15-25 watts appears to be a very large ramp increment in patients with moderate to severe COPD. Please explain why these very large numbers were chosen, and how that may have affected the findings in the study (i.e. short ramp times may decrease time for ventilatory compensation of metabolic acidosis).\n\nTable 1 – Consider including peak VO2/kg/kg/min, peak WR, peak RER, MMVV-VE (L/min), and end tidal CO2 values for the various subjects (and in the accompanying spreadsheet). The spreadsheet accompanying the article is very helpful and the authors should be commended for making the data available. A large number of subjects (~ 70%) had a VE/MVV below 85% or > 15 liters/minute (MVV-VE) at end exercise, indicating a lack of ventilatory limitation in most of these subjects.\n\nFigure 1 – As mentioned above, a large number of the subjects had no change in DVE/VCO2 or DVE/VO2. Although Table 3 addresses it, it would appear that the authors have neglected to mention this as a limitation of the entire study in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5278",
"date": "10 Mar 2020",
"name": "Samir Nusair",
"role": "Author Response",
"response": "We would like to thank Prof. Stringer for his valuable comments. This a retrospective study with small number of patients, and we certainly acknowledge the inherent significant limitations, which are referred to in the Discussion section We consider this work as a preliminary/exploratory study, and as that it has well acknowledged limitations and implications. We added the term “exploratory study” in the abstract and the introduction section to allude to that fact. We would like to address the concerns raised by the Reviewer. Re Major comments: 1. At the time of performance of these CPET studies, we did not routinely perform repeated inspiratory capacity maneuvers during exercise to allow detecting dynamic hyperinflation, because of lack of both software add-on and experience in performing theses maneuvers. Notably, dynamic hyperinflation is associated with changes in tidal volume/vital capacity ratio (Vt/VC) towards peak exercise with lower ratios of Vt/VC as dynamic hyperinflation is more evident during exercising at higher workloads. (Ref: O'Donnell DE, Revill SM, Webb KA. Dynamic hyperinflation and exercise intolerance in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2001;164:770-7) All in all there was a very little decrease in Vt/VC obtained at peak exercise, with average values of 46%+8% (Table 1). We now added to Table 3 a comparison of Vt/VC values of patients who had an increment in VE/VCO2 as opposed to patients who did not (deltaVE/VCO2=0). We found that the peak exercise Vt/VC did not differ between the two groups. Despite the limitations of this method, we conclude that dynamic hyperinflation probably did not play a key role in preventing VE/VCO2 increment from nadir values to peak exercise in our patients. The last paragraph in the Results section was altered to describe the added data and findings, and also description of findings investigating possible ventilatory mechanical constraints. Additionally, the Discussion alluded to the possible use of Vt/VC as a way to evaluate mechanical constraints on ventilation (paragraph before last). 2. Throughout the years 2010-2016 we used the same CPET and plethysmography devices described in the methods and applied the same calibration methods as recommended by the manufacturer. 3. As patients have more severe obstructive abnormality (GOLD III-IV more than GOLD I-II), they have lower ventilation, with reduced VE/VCO2 and VE/VO2 values. This has been referred to in the Introduction section, paragraph 3. Certainly, if we had bigger number of patients and all GOLD classes of patients were represented equally, we may have been able to deduce important differences between these classes. Data on end-tidal PCO2 has now been provided. 4. The fact that many patients had no increment in ventilatory equivalents from nadir to peak exercise (i.e., deltaVE/VCO2=0) is a limitation of the measurement. As mentioned in the study limitations paragraph, this may as well be explained by suboptimal effort of the patients or the ventilatory limitations of these patients. We referred to this issue now in the Limitations paragraph. 5. Data of VE/VCO2 slope was added. There was a significant correlation between Nadir VE/VCO2 and VE/VCO2 slope (R=0.82 95%CI 0.59-0.92, p<0.0001). Data of VE/VCO2 intercept was also added. (Represented in all 3 Tables). 6. We do not take arterial blood gases during routine clinical CPET performance to keep the test acceptable for our patients. This may be considered in a future prospective study if approved by ethical board. Alternatively, there are minimally invasive methods to allow blood lactate measurements which we do not have available in our clinical service. Regarding end-tidal CO2, we added the end-tidal PCO2 values at rest and at peak exercise. (See further, response to comment 7). 7. The reviewer brings our attention to the findings from a previous study which shows that COPD patients have higher arterial PCO2 and lower pH in response to blood lactate elevation during exercise, compared to people without COPD. This is somewhat expected, as with the presence of ventilatory constraints, these patients are not able to develop sufficient ventilatory compensation during exercise. In this study we did not perform these measurements; However, end-tidal PCO2 did not decrease towards peak exercise, in the patients who did not have VE/VCO2 increments, thus probably in agreement with the previously published findings highlighted by the reviewer. We added a reference to the suggested paper in our discussion. Note that at the end of the discussion (paragraph 2), we refer to the possible implication of limited ability of COPD patients of coping with metabolic acidosis from other reasons (e.g., diabetic acidosis) when they are not able to generate ventilatory compensation. Re Minor Comments: 1. We agree with the reviewer and “effort” was changed to “exercise” as suggested. 2. We agree with the reviewer regarding the importance of deconditioning as a reason for reduction in exercise capacity in COPD patients. However, the opening sentences were concentrated around describing the pulmonary mechanisms in exercise limitation in these patients. We do refer to deconditioning in the last paragraph of the Introduction. 3. Introduction, paragraph 2: “exhausted” was changed to “decreased” as suggested. 4. Citations of References 5 & 6 were joined twice in the Introduction (Paragraph 3) as suggested. 5. To identify patients and include them in the study, we looked into the CPET system software and identified all the patients that underwent CPET between the years 2010-2016 (on the same CPET device) and had a background diagnosis of COPD. Then from those patients we included only the patients that underwent body plethysmography concurrently in our laboratory, on the same day of CPET or within few days close to the date of CPET performance. 6. As stated above, there were no changes in the equipment during the 7 years. 7. Ramp increment is routinely chosen to allow for predicted WR to be achieved within 8 minutes of exercise. Only one patient exercised at a ramp of 25W/min. All other patients had a ramp of 15-20W/min. We agree with the reviewer that lower ramps may be warranted for more debilitated patients, which we often offer our patients who report low activity level. However, most patients do not handle a longer period of loaded exercise, because of other factors such as claustrophobia and mental fatigue. Regarding the effect of short ramp times on ventilatory compensation, we agree with the reviewer that higher ramps may shorten the time interval of ventilatory compensation and make it harder to evaluate the ventilation increment during the respiratory compensation phase but we doubt that this might affect the findings of our study. Perhaps, a future prospective evaluation, with exercise tests performed at lower ramps may allow a better evaluation of ventilation increment towards peak exercise. A note on that is added in study limitations. 8. We considered the suggestions of the Reviewer regarding the expanding of Table 1 to include data already shared with the readers in the accompanying research data file which is already available at data sharing site. Regarding peak VO2 in ml/min/kg, peak WR, peak RER, and MVV-VE, were added to Table 1 in the body of the article, and also in Table 3 which shows comparison between those who did not have VE/VCO2 increment and those who did. Interestingly, VO2 in ml/kg/min was found to be different between the two groups, unlike VO2 expressed as % of predicted VO2max. 9. The absence of ventilatory equivalent increment (i.e., delta VE/VCO2=0) has now been addressed in the study limitations paragraph."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1661
|
https://f1000research.com/articles/8-177/v1
|
12 Feb 19
|
{
"type": "Research Article",
"title": "Biometric and genetic differences in kelabau (Osteochilus spp.) as revealed using cytochrome c oxidase subunit 1",
"authors": [
"Nur Asiah",
"Junianto Junianto",
"Ayi Yustiati",
"Sukendi Sukendi",
"Melta Rini Fahmi",
"Zainal A. Muchlisin",
"Muhamad Kadapi",
"Nur Asiah",
"Junianto Junianto",
"Ayi Yustiati",
"Sukendi Sukendi",
"Zainal A. Muchlisin",
"Muhamad Kadapi"
],
"abstract": "Background: Kelabau (Osteochilus spp.) is a freshwater fish commonly found in the rivers of Riau, Indonesia. Researchers believe that these are Osteochilus kelabau; however, accurate taxonomic determination of these fish in Riau waters has not been made. The purpose of this study was to facilitate the identification of the kelabau based on its morphology and genetics using biometric and cytochrome c oxidase subunit 1 (CO1) analyses, respectively. Methods: Fish samples were collected from the Siak, Kampar and Rokan rivers in Riau Province, Indonesia. The DNA of 90 fish was extracted from the caudal fins using a DNA extraction kit, after which it was amplified using primers Fish-F1 and Fish-R1. Sequencing was conducted by Applied Biosystems Macrogen Korea, and the DNA sequences were then edited and aligned using MEGA v. 7. All samples were BLAST-searched for identification using the National Center for Biotechnology Information and BOLD System. Phylogenetic trees were constructed, and similarity index was calculated using accession numbers AP011385.1 and KC631202.1 in GenBank. Results: Analysis of the consensus barcode sequence for 86 species revealed a high percentage of barcode matches (96%–97% in GenBank and 96.6%–96.76% in the BOLD System). The nucleotide distance between groups of kelabau from the different rivers based on the Kimura 2-parameter model gave the following results: 0.05% between groups from the Siak and Kampar rivers, 0.09% between those from the Siak and Rokan rivers and 0.05% between those from the Kampar and Rokan rivers. The nucleotide distance between the groups in the Siak (0.09%), Kampar (0.00%) and Rokan (0.10%) Rivers indicated that the kelabau in those rivers were related to each other. Conclusions: Based on the results of the research data using CO1 and biometric analyses, the kelabau were confirmed to be O. melanopleurus.",
"keywords": [
"DNA barcoding",
"Kelabau Fish",
"Common Rivers of Riau",
"Population Structutre"
],
"content": "Introduction\n\nKelabau are ancient fish belonging to genus Osteochilus of family Cyprinidae. The species is distributed throughout Thailand, Vietnam, Peninsular Malaysia, Borneo and Sumatra1,2. In Sumatra Island in Indonesia, the fish is commonly found in the Siak, Kampar and Rokan rivers in Riau Province3,4. The demand for it as a food resource is increasing; however, its populations are decreasing in several major rivers in Sumatra, particularly in Riau Province5,6, and its cost is increasing. Several factors, such as overfishing, electrofishing and changes in river ecosystems, might play a role in the decline of the populations of these as well as many other fish5,7.\n\nAccording to local fishers in Riau, kelabau are divided into two types on the basis of morphology; however, there is no detailed information about these fish types. Thus, a study was needed to identify the species using morphological and molecular methods to determine these types in the Siak, Rokan and Kampar rivers. Identification of any species using morphological traits can be difficult and can lead to errors8; therefore, owing to morphological similarities among Osteochilus spp., molecular markers, such as DNA barcodes, are important to identify the fish species uses a specific sequence region (i.e. cytochrome c oxidase subunit 1 (CO1)) to identify a species and is a technique that can identify taxonomic units as well as biodiversity for determining species of several organisms8–12. Unlike molecular phylogeny used to determine relationships among species, the purpose of DNA barcoding is to identify unknown or undetermined species into phylogeny13. The common mitochondrial (mt) DNA region used as a barcode in protists and animals comprises 600 bp. In addition, CO1 is one of the genetic markers used to identify insects, birds, primates and fish to species14–16. MtDNA CO1 is selected as a target in DNA barcoding because it is a highly conserved site. This method has advantages over the morphological identification approach in that it is fast, reliable and it can be used for all types of samples because it uses a single gene along with mutations in the nucleotides to acknowledge the taxonomic features of each species16.\n\nThe study on DNA barcoding for freshwater fish has been widely practised in various countries, including Nigeria17, Malaysia18, Philippines19, Canada12 and Indonesia8,20–22. The method has been successfully validated for the taxonomic status within Rasbora in Lake Laut Tawar23; Anguillidae in Aceh waters24; Ornamental fish from Peat lands8; Channidae in Peninsular Malaysia, Sarawak, Sumatera, Borneo, Myanmar, Vietnam, India, Germany, Singapore and the United Kingdom25 and Cichlidae in northeastern Nigeria26; therefore, it can be used to equally successfully validate the taxonomic unit of the kelabau using its morphology supported by molecular data. This information is crucial for designing a remedial course of action with regard to the conservation strategy for this species in the Siak, Kampar and Rokan rivers in Riau Province, Indonesia.\n\n\nMethods\n\nThe study population was collected and sampled according to the guidelines on the use of living organisms for research from the Laboratory of the Faculty of Fisheries and Marine, Riau University, Indonesia.\n\nA total of 90 kelabau (30 fish from each river) were collected from the Siak, Kampar and Rokan rivers (Figure 1). Fish were caught using a gill net 3 m deep and 20 m long with a 12.7-cm mesh. The gill nets were installed in the river water close to the riverbank and remained for 24 h from 08:00 to 08:00 the following day. The fish collected were counted using handcounter and cleaned using freshwater. A number of 50-mm caudal fin tissue samples were taken using a sterile scissors and preserved in ethanol, after which a photo of each fish sample was taken for documentation using a digital camera.\n\nAll samples were preserved in 3-kg sample bags which were labelled according to site location, date and serial number. Before preservation, the fish samples were injected with 10% formalin. The fish samples were then transported to the laboratory for further evaluation. The morphologies of the collected fish were identified up to species level using the identification book produced by the Indonesian Institute of Sciences ichthyology museum2,27. The fish morphologies observed were length, colour, shape of scales, mouth shape, barbels, number of fins and special marks on the body.\n\nBiometric analyses were used to measure morphological characteristics in this study2. This tool is considered conventional for identifying organisms. Molecular identification using CO1 gene sequences has been supported for providing additional organism classification.\n\nDNA was extracted using the spin-column method from the gSYNC DNA Extrusion Kit (Geneaid Catalogue No. GS 300, Taiwan). The extracted DNA was than transferred to a 1X Tris–borate ethylenediaminetetraacetic acid (TBE) solution with a 1.5% agarose gel and Pegreen gel dye (PEQLAB Biotechnologie GmbH, Erlangen, Germany)22. The quantity of DNA was visualised with the help of a GeneQuant Spectrophotometer by adding 78 μL nuclease-free water in a cuvette along with 2 μL DNA. The DNA was then amplified using the universal primer Fish-F1 (5'-TCA-ACC-AAC-CAC-AAA-GAC-ATT-GGC-AC-3') and Fish-R1 (5'-TAG-ACT-TCT-GGG-TGG-CCA-AAG-AAT-CA-3') with a target of 707 bp and 655 bp28, respectively. The amplification thermocycling conditions as follow: the PCR condition using pra PCR (94°C for 5 min), 35 cycles of denaturation (94°C for 30 s), annealing (56.6°C for 30 s) and extension (72°C for 30 s), followed by post-PCR extension (72°C for 5 min) and hold (4°C for 5 min). PCR results were analysed using 1.5% agarose gel at 100 V to assess the bands, and only the clear products were sent to Applied Biosystems Macrogen Korea for sequencing.\n\nThe PCR amplicon was 707 bp, which implied that no sequence of DNA was derived from mtDNA nuclear mitochondrial DNA segments (NUMTs), because a NUMT barely reaches 600 bp12. The selected CO1 sequences were entered into GenBank and the BOLD System databases to compare the alignment of nucleotide sequences and 99%–100% values with that with no insetions/deletions. All sequences were aligned using ClaustalW with MEGA v.729.\n\n\nData analysis\n\nThe entire nucleotide sequence obtained from the sequence chromatogram was assembled using DNA Baser Assembler, aligned and then analysed using MEGA 7. It was further aligned (multiple alignments) using the reference NCBI GenBank accession numbers AP011385.1 and KC631202.1. Similarly, the percentage of CO1 sequences were blasted using NCBI Blast and BOLD Systems databases.\n\nNucleotide variations among samples were analysed using dnaSP v.530. The parameters of these calculations were haplotype number, variable site, parsimony site, haplotype diversity and nucleotide diversity.\n\nPhylogenetic trees were estimated using all samples from the three populations and calculated according to the Tamura-Nei model31 using MEGA 729.\n\nThe distance among the nucleotide bases of the mtDNA CO1s was analysed using the Kimura 2-parameter model32. The nucleotide distances between and within the populations were examined according to the model based on the similarity of frequencies and ratios of transition to transversion (Ti:Tv) using MEGA 729.\n\n\nResults\n\nThe morphological traits of all kelabau used in this study matched those of O. melanopleurus. We used the important morphological traits to identify these fish according to Kottelat et al.2. The morphological characteristics measurement of O. melanopleurus showed that the fish have 16–19 branched dorsal rays, the number of scales was ranged from 10.5 to12.5 in between dorsal origin and lateral line, the number of circum peduncular rows of scale was ranged from 20 to 24 and lips covered with folds and plicae and there was no hard tubule at the tip of the mouth (Figure 2a). This species has one pairs of barbels at above and one pairs at bottom, dark hazy blotches near above of the pectoral fins. The body is brownish, with the bottom brighter than the top and the type of ctenoid scales (Figure 2b). Raw biometric data are available on OSF33.\n\n(a) Kelabau (O. melanopleurus) and (b) ctenoid scale of O. melanopleurus.\n\nA sequence amplified by Fish-F1 primer was successfully identified in 86 of 90 samples of mtDNA fish. The base length of the CO1 nucleotide obtained from the formulation process and electrophoresis (Figure 3) was ~707 bp.\n\nBased on genetic analysis using the Tamura-Nei model, there was an unequal distribution of all nucleotides with the following frequencies: adenine (A), 26.73%; thymine (T), 30.44%; cytosine (C), 25.93% and guanine (G), 16.90% (Table 1). The rates ratio between transition and transversion was 10.257 purines and 1.915 pyrimidines, and the overall transition and transversion bias was R= 2.499 based on Tamura-Nei model34. The pattern of nucleotide distribution of A, T, C and G was T > A > C > G, but the carps which cultivated in India have the following pattern of nucleotide distribution: T > C > A > G35.\n\nBold: different transitional subtitutions; italic: tranversional subtitutions.\n\nThe nucleotide distances between nucleotide bases within the groups indicated that the values of the nucleotide base sequences within the fish population were 0.0009, 0.0000 and 0.0010 in the Siak, Kampar and Rokan rivers, respectively. The evolutionary distance between the nucleotides of the groups had a comparative difference in the nucleotide sequences of 0.0005 for fish from the Siak and Kampar rivers, of 0.0009 in those from the Siak and Rokan rivers and of 0.0005 for those from the Rokan and Kampar rivers. Based on the nucleotide distance, the fish were identified as being from the same species (0.06%) (Table 2).\n\nSource: Kimura estimation of 1980: “Evolutionary Divergence over Sequence Pair Between Groups”32.\n\nCO1 had 612 conserved sites (98%), 9 variable sites (1.45%), 4 informative parsimony (0.64%) and 5 singleton sites (0.81%). The highest nucleotide variation was in the Rokan river population (0.00100 ± 0.00032); whereas, the Kampar river population had no nucleotide variation based on DnaSP5 calculations (Table 3). Using the NCBI database with accession numbers AP011385.1 and KC631202.1, the DNA sequence of Kelabau was identified as belonging to O. melanopleurus with 96%–97% accuracy, query coverage of 99%–100% and an E-value of 0.0. While based on the BOLD System, the identity of all samples was 96.60%–96.93% accurate.\n\nSource: Nei, 1987 for haplotype and nucleotide diversity36.\n\nIn the phylogenetic tree consisted of two major groups (Figure 4). The first group was Cirrhinus moltonela (GenBank KC631192.1) and it was divided from O. melanopleurus. The second group was differentiated into two sub groups, O. melanopleurus from GenBank (AP011385.1 and C631202.1) and 86 fish samples from Kampar, Siak and Rokan rivers. The 86 samples have BLASTN similarity values with O. melanopleurus of 96%–97%.\n\n\nDiscussion\n\nOverall, the morphological traits and DNA barcoding showed that the majority of, if not all, kelabau fish in the three rivers at Riau Province were O. melanopleurus. Although the local fishermen have reported that there are two types of kelabau fish in the Riau river, these differences could the result of environmental changes and overfishing.\n\nEnvironmental changes can cause fish death or migration to suitable habitats. Overfishing using both legal and illegal methods has also triggered the decline of certain species5,7. In addition, our results suggested that there was little nucleotide diversity among O. melanopleurus in the Siak, Rokan and Kampar rivers in Riau Province, particularly the fish in the Kampar river\n\nThe lack nucleotide diversity of O. melanopleurus from the three rivers was likely to be caused by limited opportunities for kelabau migration, so that the genetic exchanges with other populations are very small7; morevoer, the lack nucleotide diversity is believed to be caused by inbreeding, and overfishing8,37.\n\nIn addition, Kelabau from the Siak and Rokan rivers were designated as one sub-sub group in group two (Figure 4) because both rivers are geographically connected, allowing for hybridisation, whereas, there is inbreeding of these fish in the Kampar River, which causes a nucleotide diversity value of 0.0. The same deductions were drawn from previous studies on Desmopuntius pentazona and D. rombochelatus, although the taxonomies of the two fish are different. Nevertheless, based on a genetic difference of only 0.4%, the two species were grouped into one cluster8. These are distributed throughout Asia, the Mekong and Chao Praya river basins, Peninsular Malaysia, Sumatra and Borneo1,2.\n\nThe identity of a species was derived using the morphological identification method to distinguish between species or individuals38,39. Basically, the genetic identification of a species can be done using mtDNA CO1, a more effective approach than using rRNA8,35. The nucleotide locus and mutations were used as references to conduct DNA barcoding in all fish samples16. Previous studies have identified several species using DNA barcoding, such as ornamental fish of wetlands8, wetland fish larvae39, rainbow fish40, Cyprinidae fish41, salmon and trout10 and freshwater fish12,22. Furthermore, the phylogenetics of CO1 sequences can effectively show congeneric and confamilial species12.\n\nThe phylogenetic trees cauld describe the line of biological evolution from species or organisms with a different ancestry42. Nonetheless, the results of all these species did not show a 100% undistinguishable identity. The branch length between species leading to a gap in the pairwise distance distribution is referred to as the barcoding gap in CO126. Intra-species relationships were quite high in all samples, which confirmed that kelabau (O. melanopleurus) were native in the three rivers and had the ability to adapt to changes in environmental conditions37.\n\nMoreover, the existence of inter-nucleotide patterns and distances between A, T, C and G in the chromosomes showed the characteristics and genetic signs that distinguished each of the individuals, even though they belong to the same species43. This is reinforced by referencing the phylogenetic tree made using the neighbour-joining model.\n\nThe identification of fish species is normally conducted using morphological characteristics such as dorsal fins, pelvic fins, pectoral fins, anal fins, linea lateral fins, upper linea lateral fins, lower linea lateral fins, around body fins, and caudal peduncle fins; however, in this study, we used 12 morphological traits as described by the classification system of Kottelat et al.2. These results supported the classification using biometric data that all fish in the three rivers were O. melanopleurus. The morphological characteristics were consistent with the species having a relatively large body with a standard length of 119–560 mm, lips covered with folds and plicae, no tubercles on the snout, a pair of maxillary barbels, and a pair of lower jaw barbels. The body is brownish, with the bottom brighter than the top. Dark hazy blotches near above of the pectoral fins, which is a special trait of O. melanopleurus.\n\nHowever, this method can be difficult, and molecular identification is necessary. In particular, using mtDNA CO1 was an effective approach12,20. The results from nucleotide distance data based on the Kimura 2-parameter model indicated that the nucleotide distance among the fish was short in intraspecific species using mtDNA CO137, which was supported by data showing that the percentage identity in O. melanopleurus species ranged between 96% and 97%. The Kelabau fish from the three sample sites were identified as O. melanopleurus by percentage identity, supported by an E-value of 0.0 and a 99%–100% query cover. The p-value indicated that the BLASTN results contained no errors. In addition, the low nucleotide distance values (<3%–5%) among the samples of O. melanopleurus from the Siak, Kampar and Rokan rivers, indicated that all samples were monophyletic.\n\n\nConclusion\n\nBased on our findings, we concluded that 86 of the 90 samples of kelabau from the Siak, Kampar and Rokan rivers in Riau were O. melanopleurus, as revealed by their morphological traits and the molecular analyses.\n\n\nData availability\n\nCO1 gene sequences and raw biometric data of Osteochilus melanopleurus from Riau rivers can be found on OSF.\n\nCO1 gene sequence DOI: https://doi.org/10.17605/OSF.IO/XGEZD44.\n\nRaw biometric data DOI: https://doi.org/10.17605/OSF.IO/CFGM833.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nThe raw CO1 sequence data were also deposited in GenBank and can be found under sequential accession numbers MH430827-MH430854 and MH459085-MH459142.",
"appendix": "Grant information\n\nThe study was supported by Doctor’s Dissertation Research SP DIPA-042.061.401516/2018, Ministry of Research, Technology and Higher Education (MRTHE), the Republic of Indonesia; therefore, the authors thank MRTHE for the financial collaboration support with the Research Institution and Community Service of Riau 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\nThe authors thank Neli Safrina as a staff Laboratory of Fisheries and Marine Faculty, Riau University and Gema Wahyu Dewantoro, as a staff researcher at the Laboratory of Ichtiology in the Field of Zoology, Research Center for Biology of the Indonesian Institute of Sciences (LIPI) on the identification of the Kelabau fish using morphological traits. Also, thanks to Ornamental Fish Aquaculture Research and Development Center, Ministry of Marine and Fisheries Republic of Indonesia, Depok for providing laboratory facilities.\n\n\nReferences\n\nKottelat M: The Raffles Bulletin of Zoology. 2013; 27. Reference Source\n\nKottelat M, Whitten AJ, Kartikasari SN, et al.: Ikan air tawar Indonesia bagian Barat dan Sulawesi. Periplus E Jakarta Indonesia. 1993. Reference Source\n\nPulungan CP: Ikan-ikan air tawar dari sungai Ukui, anak sungai Siak, Riau. Terubuk. 2011; 39(1): 24–32. Reference Source\n\nAsiah N, Junianto J, Yustiati A, et al.: Morfometrik dan Meristik Ikan Kelabau (Osteochilus melanopleurus) dari Sungai Kampar, Provinsi Riau. J Perikan dan Kelaut. 2018; 23(1): 47–56. Reference Source\n\nNasution S, Nuraini: Grant of Feed Containing Vitamin E in Home Fish Kelabau (Osteochilus Kelabau) to Improve Quality Eggs and Larvae. Int J Sci Eng Res. 2014; 2(4): 4–9. Reference Source\n\nAryani N: Native species in Kampar Kanan River, Riau Province Indonesia. IJFAS. 2015; 2(5): 213–217. Reference Source\n\nKusmini II, Gustiano R, Mulyasari A: Karakteristik genetik ikan Kelabau (Osteochilus kelabau) dari berbagai lokasi Kalimantan Barat menggunakan metode RAPD (Random Amplified Polymorphism DNA). Ber Biol. 2011; 10(4): 449–454. Reference Source\n\nFahmi MR, Prasetio AB, Kusumah RV, et al.: Barcoding DNA ikan Hias Lahan Gambut. Aquac Res J. 2016; 11(13): 137–145. Publisher Full Text\n\nHajibabaei M, Singer GA, Hebert PD, et al.: DDNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet. 2007; 23(4): 167–172. PubMed Abstract | Publisher Full Text\n\nRasmussen RS, Morrissey MT, Hebert PD: DNA barcoding of commercially important salmon and trout species (Oncorhynchus and Salmo) from North America. J Agric Food Chem. 2009; 57(18): 8379–8385. PubMed Abstract | Publisher Full Text\n\nTaylor HR, Harris WE: An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding. Mol Ecol Resour. 2012; 12(3): 377–388. PubMed Abstract | Publisher Full Text\n\nHubert N, Hanner R, Holm E, et al.: Identifying Canadian freshwater fishes through DNA barcodes. PLoS One. 2008; 3(6): e2490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKress WJ, Wurdack KJ, Zimmer EA, et al.: Use of DNA barcodes to identify flowering plants. Proc Natl Acad Sci U S A. 2005; 102(23): 8369–8374. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDawnay N, Ogden R, McEwing R, et al.: Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci Int. 2007; 173(1): 1–6. PubMed Abstract | Publisher Full Text\n\nJeong TJ, Jun J, Han S, et al.: DNA barcode reference data for the Korean herpetofauna and their applications. Mol Ecol Resour. 2013; 13(6): 1019–32. PubMed Abstract | Publisher Full Text\n\nHebert PD, Cywinska A, Ball SL, et al.: Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270(1512): 313–321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalade MO, Opene AJ, Benson O: DNA barcoding of Clarias gariepinus, Coptodon zillii and Sarotherodon melanotheron from Southwestern Nigeria [version 1; referees: 2 approved]. F1000Res. 2016; 5: 1268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBright AR, Sathyan N, Antony SP, et al.: Phylogeny and genetic divergence of three phenotypic variants of the ornamental goldfish, Carassius auratus (Linnaeus, 1758 ) based on CO1 gene. Int J Res Zool. 2013; 3(1): 4–9. Reference Source\n\nGuino RS II, Quilang JP, Geraldino JPL, et al.: DNA Barcoding of Philippine Mullets (Family Mugilidae: Osteochthyes). 2017; 1–23. Reference Source\n\nFahmi MR, Kusumah RV, Ardi I, et al.: DNA Barcoding Ikan Hias Introduksi. J Ris Akuakultur. 2017; 12(13): 29–40. Publisher Full Text\n\nMuchlisin ZA, Thomy Z, Fadli N, et al.: DNA Barcoding of Freshwater Fishes from Lake Laut Tawar Aceh Province, Indonesia. Acta Ichthyol Et Piscatoria. 2013; 43(1): 21–29. Publisher Full Text\n\nRosnaeni A, Elfidasari D, Fahmi MR: Dna Barcodes of the Pleco (Loricariidae, Pterygoplichthys) in thee Ciliwung River. Int J Adv Res. 2014; 5(2): 33–45. Publisher Full Text\n\nMuchlisin ZA, Fadli N, Siti-Azizah MN: Genetic variation and taxonomy of Rasbora group (Cyprinidae) from Lake Laut Tawar, Indonesia. J Ichthyol. 2012; 52(4): 284–290. Publisher Full Text\n\nMuchlisin ZA, Batubara AS, Fadli N, et al.: Assessing the species composition of tropical eels (Anguillidae) in Aceh Waters, Indonesia, with DNA barcoding gene cox1. [version 1; referees: 1 approved, 2 approved with reservations]. F1000Res. 2017; 6: 258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConte-Grand C, Britz R, Dahanukar N, et al.: Barcoding snakeheads (Teleostei, Channidae) revisited: Discovering greater species diversity and resolving perpetuated taxonomic confusions. PLoS One. 2017; 12(9): e0184017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSogbesan OA, Sanda MK, Ja’afar JN, et al.: DNA Barcoding of Tilapia Species (Pisces: Cichlidae) from North-Eastern Nigeria. J Biotechnol Biomater. 2017; 7(4): 1–4. Publisher Full Text\n\nRobert TR: The Freshwater Fishes of Western Borneo (Kalimantan Barat, Indonesia). California Academic of Sciences. San Francisco. 1989; 210. Reference Source\n\nWard RD, Zemlak TS, Innes BH, et al.: DNA Barcoding Australia’s Fish Species. Philos Trans R Soc Lond B Biol Sci. 2005; 360(1462): 1847–1857. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–1874. PubMed Abstract | Publisher Full Text\n\nLibrado P, Rozas J: DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009; 25(11): 1451–1452. PubMed Abstract | Publisher Full Text\n\nSaitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4): 406–425. PubMed Abstract | Publisher Full Text\n\nKimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–120. PubMed Abstract | Publisher Full Text\n\nAsiah N: Biometric of Kelabau Fish in Riau. 2019. http://www.doi.org/10.17605/OSF.IO/CFGM8\n\nTamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004; 101(30): 11030–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohanty M, Jayasankar P, Sahoo L, et al.: A comparative study of COI and 16 S rRNA genes for DNA barcoding of cultivable carps in India. Mitochondrial DNA. 2013; 26(1): 1–9. PubMed Abstract | Publisher Full Text\n\nNei M: Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci U S A. 1973; 70(12): 3321–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrankham R, Ballou JD, Briscoe DA, et al.: Introduction to conservation genetics. 2002. Reference Source\n\nZhang H, Yu H, Gao T, et al.: Analysis of genetic diversity and population structure of Pleuronectes yokohamae indicated by AFLP markers. Biochem Syst Ecol. 2012; 44: 102–108. Publisher Full Text\n\nNicolas H, Kadarusman AW, Frédéric B, et al.: DNA barcoding Indonesian freshwater fishes: challenges and prospects. DNA Barcodes. 2015; 3(1): 144–169. Publisher Full Text\n\nKadarusman, Hubert N, Hadiaty RK, et al.: Cryptic diversity in Indo-Australian rainbowfishes revealed by DNA barcoding: implications for conservation in a biodiversity hotspot candidate. PLoS One. 2012; 7(7): e40627. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollins RA, Armstrong KF, Meier R, et al.: Barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade. PLoS One. 2012; 7(1): e28381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPramono TB, Artiati D, Widodo MS, et al.: Identifikasi Ikan Genus Mystus Dengan Pendekatan Genetik. J Sumberd Akuatik Indopasifik. 2017; 1(2): 123–132. Publisher Full Text\n\nAfreixo V, Bastos CA, Pinho AJ, et al.: Genome analysis with inter-nucleotide distances. Bioinformatics. 2009; 25(23): 3064–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAsiah N: raw data CO1 gene in Kelabau fish. 2019. http://www.doi.org/10.17605/OSF.IO/XGEZD"
}
|
[
{
"id": "44418",
"date": "04 Mar 2019",
"name": "Indra Junaidi Zakaria",
"expertise": [
"Reviewer Expertise Fish Biology and Fisheries 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\nGenerally:\nThere are still a lot of grammatical and spelling mistakes throughout the paper. Therefore, the authors should remove all of the grammatical and typographical errors as well as spelling mistakes. Use past tense and rephrase the sentences.\n\nIntroduction:\nInconsistency in writing in the first paragraph, the first line: it is written as “genus” but in the second row it is written as “species”. Kelabau are ancient fish belonging to the genus Osteochilus of family Cyprinidaes. The species are distributed throughout Thailand, Borneo and Sumatra (etc.).\n\nAccording to local fishers in Riau, kelabau are divided into two types on the basis of morphology. Although there is no detailed information about these fish types, please, the authors should explain what the differences in the characteristics are of the two species which the fishermen know.\n\nSentences from: “The demand for it as…” until “…the population of these as well as many other fish.” I suggest deleting it, because it has no correlation with the theme of this manuscript which explains the verification of the species of fish kelabau using cytochrome c oxidase - it is not about increasing demand and the problem of decreasing populations of kelabau fish.\n\nMethods:\nCite references in the following paragraph: “The study population was collected and sampled…” until “…of the Faculty of Fisheries and Marine, Riau University, Indonesia.”\n\nResults:\nOk.\n\nDiscussion:\nThe Discussion is not up to the mark, especially in paragraphs one and two; please rewrite back with an explanation of how the characteristics of the two species of fish differ according to fishermen and the reason that these differences were caused by environmental changes and overfishing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "53842",
"date": "04 Oct 2019",
"name": "Ja’afar Nuhu Ja’afar",
"expertise": [
"Reviewer Expertise Molecular diagnostics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article needs to be proofread by an expert. There are a lot of areas I have highlighted to guide the authors.\nIn paragraph three of the discussion section, the authors should be more scientific in providing reasons for limited variation such as inbreeding within the species due to limited migration. More so, if COI is known to be conserved within a species, it is not expected to change within a small time frame of genetic event. In general, the discussion should be rewritten to explain the results and interpretations of the study.\nIn addition, I suggest the authors contact an expert that can explain/interpret the results obtained using DNASP. The explanation of the results from the software should be more than presenting the values.\nThe gel result will be more appreciable if the marker has been properly labelled.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "55212",
"date": "04 Nov 2019",
"name": "Jayasankar Pallipuram",
"expertise": [
"Reviewer Expertise I have been working on molecular taxonomy of fish",
"shellfish and marine mammals since 1996",
"thus 23 years of experience in this field",
"also gained expertise in freshwater aquaculture technology during the past 11 years."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDNA barcoding is an important and useful tool to ratify species status in circumstances where morphology-based taxonomy fails or nearly-fails to perform unambiguous identification of species. The authors in the present study have taken up an important group of freshwater fish. There are many grammatical errors and some technical flaws in the manuscript. I felt one major drawback could be their total silence on the nominal species of Osteochilus kelabu. Of course using barcoding they have proved that the species is O. melanopleurus. However, they should have shown a phylogenetic comparison of these two nominal species to rule out confusion in identification. Morphological method followed does not appear to be robust. They are advised to use TRUSS MORPHOMETRICS which can be performed using user-friendly software nowadays. In the phylogenetic tree the majority of bootstrap values do not appear to be robust; hence it could reflect a spurious relationship.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-177
|
https://f1000research.com/articles/7-1706/v1
|
26 Oct 18
|
{
"type": "Research Article",
"title": "Different drying temperatures modulate chemical and antioxidant properties of mandai cempedak (Artocarpus integer)",
"authors": [
"Anton Rahmadi",
"Yuliana Sabarina",
"Sukmiyati Agustin",
"Yuliana Sabarina",
"Sukmiyati Agustin"
],
"abstract": "Background: Mandai, the fermented inner skin of cempedak (Artocarpus integer), may have further use as industrial ingredient while maintaining its antioxidative capacity. To promote fermentation, Lactobacillus casei was induced as the starter culture. This research was carried out (i) to investigate the effect of temperature on yield, chemical properties, and antioxidant activity of starter induced fermented mandai powder, (ii) to find the best drying temperature for the powder, and (iii) to find correlations between phenolic contents and antioxidant activity of the powder. Methods: The drying temperature was used as the variable, and was set at 45, 50, and 55°C at a fixed duration of 18 hours. The control was spontaneously fermented mandai dried at 50°C for 18 hours. Total phenolic content (TPC), hydrolyzed tannic content (HTC), and total flavonoid content (TFC) were spectrophotometrically measured, expressed gallic acid (GAE), tannic acid (TAE), and catechin (CAE) equivalents. Antioxidant capacity was measured by DPPH assay. Results: The best mandai powder had total phenolic content of 348.8±55.6 mg GAE kg-1, HTC of 143.8±9.3 mg TAE kg-1, TFC of 17.5±1.3 mg CAE kg-1, antioxidant activity (IC50) of 56.96 ppm, ash content of 4.0±0.7%, pH value of 5.0±0.8, and yield of 9.3±0.8%. There was a strong correlation between TPC, HTC, TFC, and the antioxidant activity. Conclusions: Drying temperature affected all observed parameters but not ash and pH. Temperature of 45°C emerged as the best treatment to produce mandai powder from L. casei-inoculated mandai cempedak fermentation. The antioxidant activity of mandai cempedak was contributed by the phenolic components.",
"keywords": [
"Antioxidant activity",
"Artocarpus integer",
"drying temperature",
"polyphenols"
],
"content": "Introduction\n\nIn South and East Kalimantan, Indonesia, people historically consume the lactic acid bacteria (LAB)-fermented inner skin of cempedak (Artocarpus integer), traditionally termed dami or mandai1. The inner skin of cempedak can be used in functional food production, which may partly reduce agricultural waste2. Mandai may contain phenolics, flavonoids, tannins, and antioxidant activity.\n\nThe unfermented cempedak inner skin (Artocarpus integer) contains bioactive components such as phenolics, flavonoids and carotenoids3. It has antioxidant activity that is potentially higher than the flesh and seeds of cempedak. Mandai with L. casei as the starter had better antimicrobial activity against S. aureus and E. coli in comparison to spontaneous (L. plantarum) mandai4. In addition, mandai may function as a probiotic food5. While maintaining its antioxidative capacity, fermented mandai may have further use as industrial ingredient primarily as an exotic tropical flavor and a flavor enhancer.\n\nMandai powder is produced through drying process. To produce good quality mandai powder, the right drying temperature is required. Drying at the correct temperature minimizes antioxidant damage, implicating the ability to reduce free radicals will be higher, as seen in sinom beverage powder6, and cumari peppers7.\n\nThis research aims (i) to measure chemical and antioxidant properties of L. casei-fermented mandai at 45, 50, 55°C of drying temperature with constant time of drying at 18 hours, which the results are then compared with spontaneously fermented mandai and dried at 50°C for 18 hours; (ii) to find the best drying temperature on starter induced fermented mandai powder; and (iii) to find correlations between phenolic contents and antioxidant activity on starter induced fermented mandai powder.\n\n\nMethods\n\nCempedak was peeled and separated from its husk and flesh, then washed and cut into pieces. The pieces of cempedak inner skin were boiled at 100°C for 5 minutes to remove the sap. The sample was drained and then boiled once more in a sealed container at 100°C for 5 minutes to soften the texture. The sample was then cooled until the temperature was less than 40°C. L. casei strain Shirota isolated from Yakult® as starter culture were inoculated at the concentration of 2% (v/v). For spontaneous fermentation, the mandai was directly stored without inoculation. The spontaneous and inoculated mandai were stored for 2 weeks at temperature of 8±2°C to allow slow fermentation to occur. After incubation, mandai was drained and blended. The puree of mandai then was dried for 18 hours at the appropriate temperature treatment, then ground and screened with an 80-mesh sieve. All reagents and corresponding suppliers are listed in Supplementary File 1.\n\nFor the analysis of TPC, HTC, TFC, and antioxidant activity, 20 g mandai powder was dissolved in 60 ml 95% ethanol (SmartLab cat no. A1035, Indonesia) and macerated for 24 hours. Mandai was filtered through filter paper (Whatmann no. 4) and the liquid extract was dried at 50°C for 16 hours.\n\nYield was measured as the ratio of mandai powder to initial mandai cempedak (w/w), while ash contents were measured as descibed8. About 2 g of sample was diluted with distilled water to a volume of 20 ml. This mixture was homogenized and allowed to soak for 15 minutes before the pH was measured.\n\nTPC was estimated by the Folin-Ciocalteu assay and expressed as gallic acid equivalents (GAE), as described previously9,10. HTC was estimated by the Folin-Ciocalteu assay and expressed in mg kg-1 tannic acid equivalent (TAE), as described11. TFC was estimated using the aluminum chloride (AlCl3) method and expressed in mg kg-1 catechin equivalent (CAE), as described12. The antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as described13. All standards were purchased from Sigma-Aldrich.\n\nData in Table 1 except IC50 of antioxidant activity were subjected to analysis of variance (ANOVA) and the significance of the difference between the averages was determined by Fisher’s least significant difference (at α =5%, analyzed with GraphPad Prism version 6.0. Values were expressed in average ± standard deviation (SD). IC50 of antioxidant activity was produced by employing the non-linear fit of one-phase association method with GraphPad Prism version 6.0. Pearson correlation analysis was performed with Microsoft Excel 2016.\n\nYield, ash, pH, total phenolic content (TPC), hydrolyzed tannin content (HTC), total flavonoid content (TFC) are presented in average ± standard deviation.\n\nLetters after the numbers indicate the least significant difference at α=5%\n\n\nResults\n\nThe yield of mandai powder at drying temperatures of 45 and 50°C did not differ significantly. However, the yield of mandai powder dried at a temperature of 55°C was significantly different to the yield resulting from treatment temperatures of 45 and 50°C (Table 1). The yield of each treatment did not differ significantly with the yield of mandai powder with spontaneous fermented that dried at 50°C (control). A comparison between the ash content of each treatment with control showed no significant difference (Table 1). The pH values of dissolved mandai powder ranged from 4.5±0.5 to 5.3±0.2. The drying temperature did not affect the pH value of the dissolved mandai powder.\n\nThe drying temperature significantly affected the TPC of mandai powder. The highest average value of TPC in mandai powder dried at 45°C. In comparison to control, TPC of each treatment was significantly different. The drying temperatures in each treatment resulted in significantly different HTC in comparison to that of control mandai. Drying temperature of 45°C produced higher HTC than that of mandai powder dried at 50°C and 55°C. The drying temperature significantly affected the TFC of mandai powder. The TFC of mandai powder which dried at 45 and 55°C were significantly different from the control, but that of dried at 50°C was not significantly different from control.\n\nHalf-maximal inhibitory concentration (IC50) value of DPPH for each treatment was obtained through one phase association equation. The drying temperature significantly affected the antioxidant activity of mandai powder. Control mandai powder extract had the highest IC50 value, while mandai powder extract dried at 45°C had the lowest IC50 value of (Table 2). The TPC, HTC, and TFC have a strong correlation with antioxidant activity. The higher TPC, HTC, and TFC value, the higher antioxidant activity in mandai powder (Table 3).\n\nY0, (0% inhibition / without extract or Vitamin C); Plateu, maximum peak value; K, inhibition rate constant depended on concentration of Vitamin C or extract; IC50, concentration of Vitamin C or extracts inhibiting DPPH reduction by 50%.\n\n0,01-0,19 very weak correlation; 0,20-0,39 weak correlation; 0,40-0,59 moderate correlation; 0,60-0,79 strong correlation; 0,80-1,00 very strong correlation.\n\nBased on Pearson correlation analysis, there was a strong correlation between total phenolic content (r = 0.796) and total flavonoid content (r = 0.783) with antioxidant activity. The strongest correlation occurred between total tannin content (r = 0.910) and antioxidant activity in mandai powder.\n\n\nDiscussion\n\nMore water in mandai evaporated at higher temperatures. This caused the yield of mandai powder to reduce with increasing drying temperature. A previous report documents that the yield may decrease with increased drying temperatures14. The drying temperature did not affect the ash content of mandai powder. The mineral resources in the spontaneous and L. casei-fermented mandai were equally used by microbes, so it did not have a different effect to ash content.\n\nDuring mandai fermentation, population of LAB increased until day 14, thus lowering pH1,5. Inferring from previous research, it was deduced that organic acids such as lactic acid and acetic acid were produced to lower the pH and caused more acidic environment on day 12 of fermentation5. The fermentation medium, duration of fermentation, and the use of starter cultures may play a role in the final pH and organic acid contents of LAB-fermented products. The previous research stated that the pH of spontaneously fermented rye dough was higher than the pH of starter-fermented rye dough15. However, cucumber pickle fermented with LAB produced organic acids that were higher than that of spontaneous fermentation16.\n\nTPC of the inner skin of cempedak was at 21.29 mg GAE kg-13. TPC of mandai powder ranged from 199.2±13.4 to 348.8±55.6 mg GAE kg-1, higher than that of unfermented cempedak. Phenolic compounds were sensitive to heat treatment, so that the drying process reduced the TPC17. The TPC of mandai powder dried at 55°C was the lowest value observed when compared to other treatments. The drying process, especially at higher temperature (i.e. 55°C), combined with the long drying time duration (i.e. 18 hours) resulted in loss of antioxidant activity18.\n\nIn dry conditions, components in the cell, such as membranes and organelles, clump together, resulting in fewer extracted phenolic compounds14. Drying at 50°C quickly disabled the oxidation of polyphenols. However, the initial oxidation of polyphenols might have occurred prior to drying and led to polyphenol degradation. Phenolic compounds are sensitive, unstable and susceptible to degradation by oxygen and light19. The enzymatic oxidation of polyphenols components is mostly caused by polyphenol oxidase7. Injury to the cell membrane liberates and therefore activates these enzymes, which convert phenolic compounds to quinones.\n\nPhenolic contents are often subjected to heat processing, such as drying, boiling, and steaming19. Environmental factors affecting phenolic concentrations include weather conditions, seasons, and post-harvest conditions20. Phenolic contents are also related to varieties of different fruits, diversity of extraction methods21, and the type of phenolic components in the plant and its location in the cell, as well as the type of solvent and method of extraction22–24.\n\nL. casei may modify the phenolic component, causing a significant difference between TPC of each treatment and control25,26. The type of fermentation and metabolic activity of LAB may play a role on levels of total phenolic in rye dough and bread15 and Moringa oleifera leaf powder27. HTC was inversely correlated with drying temperature. In addition, duration of drying contributes to the loss of tannins28,29. This is consistent with the results in yacon (Polymnia sonchifolia) and coffee leaf tea30,31. Degradation of flavonoid structures is linked to the degree of heat exposure30,32.\n\nThe antioxidant activity of mandai powder was low when compared to the fresh form. Temperature plays a role in retaining antioxidant activity of the powder (Table 2). Temperature had a significant effect on the inhibition of free radicals of DPPH in grass jelly (Premna serratifolia)33. A strong correlation was observed between antioxidant activity and the polyphenol contents of mandai powder. Previous studies have reported strong correlations between TPC, HTC, TFC and antioxidant activity34,35. Phenolic chemical structure has a role in the inhibition of free radicals, largely depending on the number and position of the hydrogen donation from the hydroxyl group to the aromatic ring of the phenol molecules36.\n\n\nConclusions\n\nDrying temperature affected total phenolic, tannin and flavonoid contents, antioxidant activity, and yield but did not affect ash content and pH of starter induced fermented mandai powder. Mandai powder dried at 45°C for 18 hours emerged as the best treatment, with a TPC of 358.8±55.6 mg GAE kg-1 dry sample, HTC of 143.8±9.3 mg TAE kg-1 dry sample, TFC of 17.5±1.3 mg CAE kg-1 dry sample, antioxidant activity (IC50) of 56.96 ppm, ash content of 4.0±0.7%, pH value of 5.0±0.8, and yield of 9.3±0.8%. The strongest correlation was shown between HTC and IC50 of antioxidant activity on starter induced fermented mandai powder. The antioxidant activity of mandai cempedak was contributed by the phenolic components.\n\n\nData availability\n\nDataset 1. All raw data obtained in the present study. Data include yield, ash weight, pH, and phenolic, tannin and flavonoid contents. DOI: https://doi.org/10.5256/f1000research.16617.d22191137.",
"appendix": "Grant information\n\nThe principal investigator would like to thank the Indonesian Ministry of Research, Technology, and Higher Education that had funded this research with contract number 128/UN17.41/KL/2018.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Nur Aini Haryati and Alamsyah for proof-reading this article.\n\n\nSupplementary material\n\nSupplementary File 1. List of all reagents used in the current study, with suppliers.\n\nClick here to access the data\n\n\nReferences\n\nNur HS: Microbial succession and biochemical aspects of mandai fermentation at low salt concentration. Makara Sains. 2009; 13(1): 13–16. Reference Source\n\nTeh SS, Ahmad R, Wan-Abdullah WN, et al.: Evaluation of agrowastes as immobilizers for probiotics in soy milk. J Agric Food Chem. 2009; 57(21): 10187–10198. PubMed Abstract | Publisher Full Text\n\nBakar MFA, Karim FA, Perisamy E: Comparison of phytochemicals and antioxidant properties of different fruit parts of selected Artocarpus species from Sabah, Malaysia. Sains Malays. 2015; 44(3): 355–363. Publisher Full Text\n\nRahmadi A, Abdiah I, Sukarno MD, et al.: Physicochemical and antibacterial characteristics of virgin coconut oil fermented with lactic acid bacteria. Jurnal Teknolologi dan Industri Pangan. 2013; 24(2): 178–183. Publisher Full Text\n\nEmmawati A, Laksmi BS, Nuraida L, et al.: Characterization of lactic acid bacteria isolates from mandai function as probiotic. Agritech. 2015; 35(2): 146–155. Publisher Full Text\n\nHartiati A, Mulyani S: The effect of maltodextrin concentration and drying temperature to antioxidant content of sinom beverage powder. Agric Agric Sci Proc. 2015; 3: 231–234. Publisher Full Text\n\nReis RC, Castro VC, Devilla IA, et al.: Effect of drying temperature on the nutritional and antioxidant qualities of cumari peppers from pará (Capsicum chinense Jacqui). Braz J Chem Eng. 2013; 30(2): 337–343. Publisher Full Text\n\nAndarwulan N, Kusnandar F, Herawati D: Food Analysis. Jakarta: Dian Rakyat. 2011.\n\nMu’nisa A, Wresdiyati T, Kusumorini N, et al.: Antioxidant activity of clove leaf extract. Jurnal Veteriner. 2012; 13(3): 272–277. Reference Source\n\nNurhayati N, Siadi K, Harjono H: Effect of sodium benzoate concentration and storage duration on total phenolic content of tomato paste. Indo J Chem Sci. 2012; 1(2): 158–163. Reference Source\n\nMalangngi LP, Sangi MS, Paedong JJE: Determination of tannins content and antioxidant activity of advocado seed fruit extracts (Persea americana Mill.). Jurnal MIPA Unsrat Online. 2012; 1(1): 5–10. Reference Source\n\nZou Y, Lu Y, Wei D: Antioxidant activity of a flavonoid-rich extract of Hypericum perforatum L. in vitro. J Agric Food Chem. 2004; 52(16): 5032–5039. PubMed Abstract | Publisher Full Text\n\nFarhan H, Rammal H, Hijazi A, et al.: In vitro antioxidant activity of ethanolic and aqueous extracts from crude Malva parviflora L. Grown in Lebanon. Asian J Pharm Clin Res. 2012; 5(3): 234–238. Reference Source\n\nHusni A, Putra DR, Lelana LYB: Antioxidant activity of Padina sp. at various temperatures and drying time. Jurnal Pengolahan dan Bioteknologi Hasil Perikanan. 2014; 9(2): 165–173.\n\nBanu I, Vasilean I, Aprodu I: Effect of lactic fermentation on antioxidant capacity of rye sourdough and bread. Food Technology Research. 2010; 16(6): 571–576. Publisher Full Text\n\nXiaoyi J, Yuan W, Xingzhu W, et al.: Effects of lactic acid bacteria inoculated fermentation on pickled cucumbers. Adv J Food Sci Technol. 2013; 5(12): 1610–1617. Publisher Full Text\n\nMasduqi AF, Izzati M, Prihastanti E: The effect of drying methods to the chemical in seaweed (Sargassum polycystum). Buletin Anatomi dan Fisiologi. 2014; 22(1): 1–9. Reference Source\n\nYoussef KM, Mokhtar SM: Effect of drying methods on the antioxidant capacity, color and phytochemicals of Portulaca oleracea L. leaves. J Nutr Food Sci. 2014; 4(6): 1–6. Publisher Full Text\n\nYuliawaty ST, Susanto WH: Effect of drying time and matodextrine concentration to physiochemical and organoleptic characteristic of Morinda citrifolia L. leaf instant drink. Jurnal Pangan dan Agroindustri. 2015; 3(1): 41–52. Reference Source\n\nJihène L, Amira T, Saber C, et al.: impact of infra-red drying temperature on total phenolic and flavonoid contents, on antioxidant and antibacterial activities of ginger (Zingiber officinale Roscoe). J Environ Sci Toxicol Food Technol. 2013; 6(5): 38–46. Publisher Full Text\n\nShofian NM, Hamid AA, Osman A, et al.: Effect of freeze-drying on the antioxidant compounds and antioxidant activity of selected tropical fruits. Int J Mol Sci. 2011; 12(7): 4678–4692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnnamalai A, Ponmari G, Sathishkumar R, et al.: Effect of drying treatment on the contents of antioxidants in Cardiospermum halicacabum Linn. Int J Pharma Bio Sci. 2011; 2(1): 304–313. Reference Source\n\nRafsanjani MK, Putri WD: Characteristic of pamello fruit peel extract using ultrasonic bath (study of solvent and extraction time). Jurnal Pangan dan Agroindustri. 2015; 3(4): 1473–1480. Reference Source\n\nRebaya A, Belghith SI, Baghdikian B, et al.: Total phenolic, total flavonoid, tannin content, and antioxidant capacity of Halimium halimifolium (cistaceae). Journal of Applied Pharmaceutical Science. 2014; 5(1): 052–057. Publisher Full Text\n\nRodríguez H, Curiel JA, Landete JM, et al.: Food phenolics and lactic acid bacteria. Int J Food Microbiol. 2009; 132(2–3): 79–90. PubMed Abstract | Publisher Full Text\n\nHur SJ, Lee SY, Kim YC, et al.: Effect of fermentation on the antioxidant activity in plant-based foods. Food Chem. 2014; 160: 346–356. PubMed Abstract | Publisher Full Text\n\nIjarotimi OS, Fagbemi TN, Osundahunsi OF: Comparative study of nutrituonal profiles and phytochemical components of raw blanched and fermented flour from the leaves of Moringa oleifera Lam. Mal J Nutr. 2013; 19(3): 371–382. Reference Source\n\nKatno Kusumadewi AP, Sutjipto: The study of drying period to tannin content on Guazama ulmifolia Lamk. Jurnal Tumbuhan Obat Indonesia. 2008; 1(1): 38–46. Reference Source\n\nNazarni R, Purnama D, Umar S, et al.: The effect of fermentation on total phenolic, flavonoid and tannin content and its relation to antibacterial activity in jaruk tigarun (Crataeva nurvala, Buch HAM). International Food Research Journal. 2016; 23(1): 309–315. Reference Source\n\nDuro HL, Hurtada WA, Rodriguez FM: Functional components and sensory evaluation of yacon (Polymnia sonchifolia L.) chips at various drying conditions. The Philippine Agricultural Scientist. 2012; 95(3): 300–304. Reference Source\n\nSiringoringo FHT, Lubi Z, Nainggolan RJ: Study of tea making from coffee leaves. J Rekayasa Pangan dan Pert. 2012; 1(1): 1–5. Reference Source\n\nSharma K, Ko EY, Assefa AD, et al.: Temperature-dependent studies on the total phenolics, flavonoids, antioxidant activities, and sugar content in six onion varieties. J Food Drug Anal. 2015; 23(2): 243–252. PubMed Abstract | Publisher Full Text\n\nChua IYP, King PJH, Ong KH, et al.: Influence of light intensity and temperature on antioxidant activity in Premna serratifolia L. J Soil Sci Plant Nut. 2015; 15(3): 605–614. Publisher Full Text\n\nSah SY, Sia CM, Chang SK, et al.: Antioxidant capacity and total phenolic content of lemongrass (Cymbopogon citratus) leave. Annals Food Science and Technology. 2012; 13(2): 150–155. Reference Source\n\nPujimulyani D, Raharjo S, Marsono Y, et al.: The effect of size reduction and preparation duration on the antioxidant activity of white saffron (Curcuma mangga Val.). J Food Pharm Sci. 2013; 1: 18–21. Reference Source\n\nAmjad L, Shafighi M: Evaluation of antioxidant activity, phenolic and flavonoid content in Punica granatum. var. Isfahan malas flowers. Intl J Agri Crop Sci. 2013; 5(10): 1133–1139. Reference Source\n\nRahmadi A, Sabarina Y, Agustin S: Dataset 1 in: Different drying temperatures modulate chemical and antioxidant properties of mandai cempedak (Artocarpus integer). F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16617.d221911"
}
|
[
{
"id": "41487",
"date": "13 Dec 2018",
"name": "Syed Ziaur Rahman",
"expertise": [
"Reviewer Expertise Pharmacology",
"Nutraceuticals"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very interesting research which has tried to explore the use of Mandai as industrial ingredient while maintaining its antioxidant activity. However, there are some suggestions and queries, which needs to be corrected along with grammatical errors.\n\nAbstract: Better, if is structured like introduction, aims and objectives, material and methods, results and conclusion. Methods: On page 3, it has been mentioned that cempedak was boiled two times at 100°c for 5 minutes to remove sap and to soften its texture, then there might be a possibility that at this temperature of 100°c, flavonoid and retinoid content be lost or decreased. The mandai storage temp, and the drying period of 18 hours, are selected but without proper referencing. Please add reference if any Extraction process: Here the liquid extract was dried at 50°c for 16 hours. Please add reference if any. Methods: There should be a mentioning of temperatures. Nowhere, temperature of 45, 50 and 55°c is mentioned, though the same is provided in the table. Results: The values mentioned on page no. 4 and in table 1 are not matching. The yield mentioned in table at 50 and 55°c are approximately same, but in the text it is mentioned that there was a significant difference. There should be proper mentioning in text under result section whatever is shown in table 1. With increase of drying temperature, there is decrease in TPC, HTC and TFC. There might be a chance to get more flavonoid and retinoid content in further lowering temperatures. Antioxidant activity results/values shown on page 4 and in table 1 are not matching. In the text it is mentioned that higher TPC, HTC, TFC value means higher the antioxidant activity, but in table, as the temperature is increasing the values for TPC, HTC and TFC are decreasing, and antioxidant activity increasing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5234",
"date": "10 Mar 2020",
"name": "anton rahmadi",
"role": "Author Response",
"response": "Addressing Comment (1) Abstract structure is assigned by the journal already. Addressing Comment (2) Two consecutive boilings at 100°C for 5 minutes assisted removal of sap in the inner skin of cempedak and texture softening. This two-step boilings can be replaced by one-step boiling at a longer time. However, the use of two consecutive boilings is necessary to reduce the length of heat exposure; therefore, this was included as an essential preparation step in our patent application on mandai fermentation. Addressing Comment (3, 4, and 5) The changes are incorporated in the second version of the article. The drying time of the mandai powder and the liquid extract were opted from the result of the preliminary optimation research, subjected to our dryer capability. Addressing Comment (6 and 7) We agree with the reviewers suggestion, i.e. (1) the yields of mandai powder at drying temperatures of 45, 50, and 55 °C did not differ significantly, (2) Lowering the drying temperature may additionally preserve the content of polyphenols, therefore, may further increase the antioxidant capacity. Addressing Comment (8) In response to the reviewer's comment no.8, we made clear that In the concept of IC 50 value, the lower the IC 50 value means the substance is more potent or showing a better antioxidant capacity."
}
]
},
{
"id": "42635",
"date": "11 Feb 2019",
"name": "Francisc Vasile Dulf",
"expertise": [
"Reviewer Expertise biochemistry",
"fermentation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 aims: to measure chemical and antioxidant properties of L. casei-fermented mandai at different drying temperatures with constant time of drying at 18 hours. The results are then compared with spontaneously fermented mandai and dried at 50°C for 18 hours. The paper proposed also to find the best drying temperature on starter induced fermented mandai powder; and to find correlations between phenolic contents and antioxidant activity on starter induced fermented mandai powder.\nThe article is well written. Data is interpreted statistically. They are compared to literature data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/7-1706
|
https://f1000research.com/articles/9-172/v1
|
09 Mar 20
|
{
"type": "Brief Report",
"title": "Findings of a feasibility study of pre-operative pulmonary rehabilitation to reduce post-operative pulmonary complications in people with chronic obstructive pulmonary disease scheduled for major abdominal surgery",
"authors": [
"Lucy L. Marlow",
"Angeline H.Y. Lee",
"Emma Hedley",
"Michael P. Grocott",
"Michael C. Steiner",
"J. Duncan Young",
"Najib M. Rahman",
"Christopher P. Snowden",
"Kyle T.S. Pattinson",
"Lucy L. Marlow",
"Angeline H.Y. Lee",
"Emma Hedley",
"Michael P. Grocott",
"Michael C. Steiner",
"J. Duncan Young",
"Najib M. Rahman",
"Christopher P. Snowden"
],
"abstract": "Background: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk of complications and death following surgery. Pulmonary complications are particularly prominent. Pulmonary rehabilitation is a course of physical exercise and education that helps people with COPD manage their condition. Although proven to improve health outcomes in patients with stable COPD, it has never been formally tested as a pre-surgical intervention in patients scheduled for non-cardiothoracic surgery. If a beneficial effect were to be demonstrated, pulmonary rehabilitation for pre-surgical patients with COPD might be rapidly implemented across the National Health Service, as pulmonary rehabilitation courses are already well established across much of the United Kingdom (UK). Methods: We performed a feasibility study to test study procedures and barriers to identification and recruitment to a randomised controlled trial testing whether pulmonary rehabilitation, delivered before major abdominal surgery in a population of people with COPD, would reduce the incidence of post-operative pulmonary complications. This study was run in two UK centres (Oxford and Newcastle upon Tyne). Results: We determined that a full randomised controlled trial would not be feasible, due to failure to identify and recruit participants. We identified an unmet need to identify more effectively patients with COPD earlier in the surgical pathway. Service evaluations suggested that barriers to identification and recruitment would likely be the same across other UK hospitals. Conclusions: Although pulmonary rehabilitation is a potentially beneficial intervention to prevent post-operative pulmonary complications, a randomised controlled trial is unlikely to recruit sufficient participants to answer our study question conclusively at the present time, when spirometry is not automatically conducted in all patients planned for surgery. As pulmonary rehabilitation is a recommended treatment for all people with COPD, alternative study methods combined with earlier identification of candidate patients in the surgical pathway should be considered. Trial registration: ISRCTN29696295, 31/08/2017",
"keywords": [
"pulmonary rehabilitation",
"respiratory"
],
"content": "Introduction\n\nIn the United Kingdom, chronic obstructive pulmonary disease (COPD) affects approximately 3.7 million people1, is responsible for approximately 30,000 deaths per year, and is the fifth most common cause of death2. COPD is an independent risk factor for postoperative complications (odds ratio OR 1.35 (CI 1.30-1.40)) and death (OR 1.29 (CI 1.19-1.39))3–6. Complications include pulmonary and cardiac events, sepsis, renal insufficiency and an increased reoperation rate3. Surgical patients with COPD thus represent a high-risk group in whom there is an unmet need to improve post-operative outcomes.\n\nPulmonary rehabilitation is “A physical exercise and education programme, tailored for each person. It includes information on looking after the body and lungs, advice on managing symptoms, including feeling short of breath, nutrition and psychological support. People who smoke are given advice on how to stop.\"7\n\nPulmonary rehabilitation is usually delivered in an outpatient setting, consisting of one hour of exercise and one hour of education, twice weekly for six weeks. It has profound benefits on breathlessness, exercise capacity and quality of life (number needed to treat (NNT)=2)8, no side effects are reported9. Pulmonary rehabilitation is associated with decreased hospital admissions (NNT=3–4), and mortality (NNT =~6) following COPD exacerbations10–13. Crucially, pulmonary rehabilitation is inexpensive9. Its effect is so powerful that it has a negative cost per quality adjusted life year (QALY), meaning it saves money for the NHS14. The main challenges facing pulmonary rehabilitation are the barriers to its uptake, as attendance and completion of the programme is often poor15,16.\n\nDespite adoption in the National Institute for Health and Care Excellence (NICE) guidelines17 for stable COPD, pulmonary rehabilitation is not regularly offered to pre-surgical patients with COPD18. We believe that pulmonary rehabilitation merits investigation as a potential means to improve postoperative outcome in people with COPD undergoing surgery for the following reasons:\n\nA handful of small surgical studies suggest beneficial effects of pulmonary rehabilitation on the incidence of postoperative pulmonary complications19–23. Differing endpoints, small sample sizes and restriction to specific surgical groups limits conclusive interpretation.\n\nIn the National Emphysema Treatment Trial (NETT)24,25, lung volume reduction surgery was compared with medical management of COPD. All patients underwent pre-operative pulmonary rehabilitation. In the thoracic surgical population of NETT similar outcomes (in terms of functional exercise capacity and health related quality of life, assessed prior to surgery) were observed to what would be expected in the treatment of non-surgical patients with COPD. In fact, approximately 10% of participants in NETT decided against lung volume reduction surgery because they felt so much better after pulmonary rehabilitation.\n\nShortened durations of pulmonary rehabilitation are efficacious8,26. This is important, because an adapted course may be necessary to fit within surgical time frames.\n\nPulmonary rehabilitation is widely available and standardised across the NHS in over 200 UK centres. This has important implications for scalability.\n\nPre-operative pulmonary rehabilitation needs sufficient time between the decision to operate and the operation, requires cross specialty working, and involves patients with two conditions (COPD and a surgical condition). A randomised controlled trial is therefore justified, as the current evidence base is either not specific to a surgical population or is case series based and therefore subject to selection bias. Furthermore, it is unclear whether a randomised controlled trial of pulmonary rehabilitation before surgery would be practical. This study investigated the feasibility of running such a large randomised controlled trial. The feasibility study design matched the expected full study design except in scale.\n\n\nMethods\n\nThis feasibility study was run as an open, parallel group, randomised trial with an allocation ratio of 1:1. The study was conducted across two research sites (Oxford and Newcastle upon Tyne), chosen as two areas with different demographics and incidence rates of COPD. Ethical approval was granted by the South Yorkshire Research Ethics Committee (approval number 17/YH/0220). Written informed consent was obtained from all participants prior to the start of the study. The primary aim of the study was to determine feasibility for a randomised controlled trial and focused on recruitment rate, barriers to recruitment and uptake of pulmonary rehabilitation. The trial was registered on ISRCTN on 19 August 2017 (ISRCTN29696295).\n\nInclusion criteria:\n\nAdult patients aged 18 years or older with COPD\n\nHas capacity to take part in this study\n\nScheduled for elective major (body cavity) surgery OR laparascopic surgery that is anticipated to last longer than 2 hours\n\nPeople with more than 20 pack years smoking history were approached to take part in the study if spirometry subsequently confirmed COPD.\n\nExclusion criteria\n\nInability to give informed consent\n\nInsufficient command of English to understand the study documentation\n\nUnable to participate in pulmonary rehabilitation treatment according to British Thoracic Society guidelines27.\n\nPatients scheduled cardiac, thoracic and orthopaedic surgery and orthopaedic surgery\n\nPatient involvement in study design: As we anticipated that recruitment to this study may be challenging, we discussed the study design with patient groups consisting of people with COPD who had either undergone surgery, or those who had experienced pulmonary rehabilitation. The key messages from these patient representatives were to ensure that transport to and from pulmonary rehabilitation would be provided, and that a flexible approach to scheduling would be necessary so pulmonary rehabilitation could fit with other appointments.\n\nParticipant identification and recruitment: To determine the best point in the surgical pathway to recruit participants, research nurses screened for study participants from the following sources.\n\nFrom the surgical multidisciplinary team (MDT) meetings\n\nIn oncology clinics\n\nFrom the electronic patient record for patients scheduled for surgery\n\nFrom hospital anaesthetic preoperative assessment clinics\n\nFrom cardiopulmonary exercise testing clinics\n\nParticipants were initially approached by their clinical team, and in those who agreed to take part in the study informed consent was taken at the first research visit.\n\nThe study aimed to collect 48 full data sets (24 in each centre, 12 pulmonary rehabilitation, 12 control arm). To achieve this 48 dataset target, based on known drop-out rates from pulmonary rehabilitation18 and potential further data loss due to surgical scheduling, we anticipated that we would need to recruit 72 patients. This sample size was chosen pragmatically, with the aim to test efficacy of recruitment, randomisation, how best deliver a control arm the best way to conduct the study across multiple sites and importantly to enable us to evaluate the performance of the primary outcome measures (e.g. for ceiling and floor effects).\n\nPulmonary rehabilitation: A pragmatic, exploratory approach was used to explore what is practically deliverable and tolerated by patients, working closely with local pulmonary rehabilitation teams in Oxford and Newcastle upon Tyne. The aim was for patients to be enrolled in 3 pulmonary rehabilitation sessions per week, for 3 or 4 weeks, depending on timing of surgery. Pulmonary rehabilitation of this shortened duration has been shown to be effective26. Patients were to attend standard NHS pulmonary rehabilitation groups run for patients with COPD.\n\nControl arm: Patients randomised to the control arm would receive standard care including advice on smoking cessation, exercise and appropriate referral and education for those with newly diagnosed COPD.\n\n\nResearch assessments\n\nRecruited participants were randomised 1:1 to either pulmonary rehabilitation or treatment as normal, minimised for study site and Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage, using an online randomisation service (Sealed Envelope Limited, London, UK). Participants were assessed prior to pulmonary rehabilitation or control treatment (preoperative assessment) and following surgery during hospital inpatient stay on postoperative days 3, 5, and 8 and again at a 6-week and 6-month follow-up (Figure 1 and below). Other than a 6-minute walk test, the research data consisted of self-report questionnaires on mood, symptoms, and quality of life. Data obtained from the NHS clinical record included various perioperative risk scores, co-morbidity scores, and measures relating to the operation and outcomes.\n\nData in yellow boxes is research data collected from the patient, whereas the data in pink relates to that collected from the patients' clinical record. Abbreviations: NHS; National Health Service, D12; Dyspnoea-12 questionnaire, CAT; COPD assessment test, QOR-15; quality of recovery score, WHODAS; World Health Organisation (WHO) disability score, STAI; Spielberger state and trait anxiety inventory, CESD; Center for epidemiolgic studies depression scale, P-POSSUM; Portsmouth Physiological and Operative Severity Score for the enumeration of Mortality, Charlston; Charlston Morbidity Index, ASA; American Society of Anesthesiologists Physical Status Classification System, Barthel; Barthel scale, Clavien-Dindo; The Clavien-Dindo Classification of surgical complications, ICU; intensive care unit, CPET; cardiopulmonary exercise test.\n\nMedical, surgical, anaesthetic assessment including comorbidities including full detailed smoking histories.\n\nPhysiology\n\n○ Spirometry\n\n○ 6 minute walk test\n\n○ Cardiopulmonary exercise testing (CPET)\n\n○ Physical activity monitoring (accelerometry-based wristwatch) – monitored for one week.\n\n○ Preoperative risk assessment scoring using POSSUM-R, Charlston Co-morbidity Index, ASA grade.\n\nPsychology and health-related quality of life\n\n○ Dyspnoea questionnaires (Dyspnea-12 questionnaire)\n\n○ Anxiety (State and Trait Anxiety Inventory), Depression (Center for Epidemiological Studies Depression Scale), Fatigue (Fatigue Severity Scale), COPD Assessment Test (CAT)\n\n○ Health status assessment with EQ-5D-5L and WHO disability assessment schedule.\n\nThese measures will be collected on postoperative days 3, 5, and 8 during hospital inpatient stay.\n\n○ Surgical factors (duration of operation, blood loss), measured once only\n\n○ Time to mobilisation\n\n○ Assessment of activities of daily living (Barthel).\n\n○ Intensive care admission, discharge, mortality\n\n○ Patient-related outcome measures, including time to return to normal activities.\n\n○ Morbidity tracking using the postoperative morbidity survey instrument and Clavien-Dindo surgical complication score.\n\nPostoperative measures collected on day 5 post-surgery only\n\n○ Health status questionnaires: Dyspnea, Anxiety, Depression, CAT EQ-5D-5L and WHO disability assessment schedule\n\n○ Smoking history\n\nData collected at discharge from hospital\n\n○ Date of discharge (i.e. length of hospital stay)\n\n○ Destination of discharge\n\nPostoperative measures collected during 6-week follow-up visit.\n\n○ Health status questionnaires: Dyspnea, Anxiety, Depression, CAT EQ-5D-5L and WHO disability assessment schedule\n\n○ Readmissions to hospital, morbidity tracking as above (from medical record).\n\n○ Smoking history\n\nPostoperative measures collected 6 months following surgery\n\nFollowing confirmation that patient remains alive (NHS Spine and communication with general practitioner) we will invite the patient to attend a follow up assessment and collect the following measures.\n\n○ Health status questionnaires: Dyspnea, Anxiety, Depression, CAT EQ-5D-5L and WHO disability assessment schedule\n\n○ Readmissions to hospital, morbidity tracking as above (from medical record).\n\n○ Smoking history\n\n\nFeasibility measures collected throughout the study\n\nCan we recruit at a sufficient rate to run an RCT?\n\nWhat is severity (GOLD/MRC) of the recruited patients and how does this compare with the screened patients\n\nWhether it is feasible to deliver the pulmonary rehabilitation intervention in the time available. This will include assess the impact of changing surgical dates, e.g. earlier (so insufficient rehabilitation delivered), later (so effect of rehabilitation wearing off).\n\nNumber of complete data sets collected\n\nPercentage of missing data\n\nBarriers to uptake of pulmonary rehabilitation\n\nNumber of patients identified in clinic with spirometry defined COPD, and their MRC and GOLD scores\n\nNumber of patients invited to participate in the study, and their MRC and GOLD scores\n\nNumber who accept invitation\n\nNumber who decline invitation but agree to participate in qualitative study\n\nNumber who decline invitation/don't reply\n\nNumber of patients who attend research assessment\n\nNumber of patients who sign consent form\n\nNumber of patients who complete pulmonary rehab or control treatment (i.e. compliance with study intervention)\n\nNumber of patients who have surgery in allocated timeframe (3 months following the second research visit)\n\nNumber of patients who continue the study during postoperative period\n\nNumber of patients in whom we can collect 6-month follow up data.\n\nScheduling of research appointments within suitable timeframes\n\nScheduling of pulmonary rehabilitation sessions within the surgical waiting time\n\nEffectiveness of transport to/from pulmonary rehabilitation. Although we plan to contribute transport costs for the study there needs to be consideration for when pulmonary rehabilitation is offered as a treatment.\n\nFactors relating to scheduling, including effect of changes in operation date.\n\nFeasibility of tracking patients postoperatively-either in person and/or via electronic and paper based patient records\n\nOutcome measures being collected to get an estimate of\n\nCentral tendency\n\nSpread\n\nData loss\n\nLoss to follow up\n\nEvent rate of postoperative complications, to help with sample size calculation for main study\n\nCheck for post randomisation dropouts because of allocation to unfavoured treatment group\n\nDo the \"treatment as normal\" patients seek exercise sessions elsewhere?\n\nIs the drop-out rate from the study similar in both groups?\n\nDoes the randomisation system work?\n\nHealth economics. We know there is health economic benefit for pulmonary rehabilitation in the treatment of COPD - does this translate to a surgical population?\n\nEQ-5D-5L measured at baseline, at day 5, 6 weeks and 6 months post operatively\n\nResource use will be measured, including primary care, pulmonary rehabilitation, hospital services(e.g. before during and after surgery)\n\nAssessors will be blinded to treatment group.\n\nWe will trial ways to ensure that the outcomes chosen are as fair as possible and are collected in a way that avoids bias. This will include objective criteria scoring by blinded individuals.\n\nOutcome measures: We anticipated that the primary research outcome measures for a future randomised controlled trial would be morbidity, mortality, length of hospital stay and hospital readmissions so we collected data on this to help with future study design.\n\nThe progression criteria to a substantive study were as follows:\n\na) Recruitment rate greater than 66% of predicted for the present study - this would account for a minimum rate that would result in a practical number of centres for a randomised controlled trial.\n\nb) Screened and recruited patients similar in severity - assessed by clinical judgment\n\nc) Compliance with treatment approximately in line with national COPD audit figures\n\nd) To be confident that there were no insurmountable barriers to the uptake and running of pulmonary rehabilitation for these patients.\n\n\nResults\n\nRecruitment commenced in Oxford in January 2018 and in Newcastle upon Tyne in May 2018. A total of 266 patients were screened of which 65 met the inclusion criteria. As of January 2019, one participant had been recruited in Oxford and two in Newcastle upon Tyne. At this point it was determined that running a randomised controlled trial of pulmonary rehabilitation would not be feasible and the study was terminated in March 2019. Further details are presented in Figure 2. We have not presented the research data here due to interpretability and confidentiality issues arising from only acquiring two datasets.\n\nWe found that the main barrier to study recruitment in both centres was associated with the way the surgical pathway is organised, especially with regards to two specific aspects; surgical timelines and identification COPD.\n\nIn Oxford, challenges were faced in identifying patients with COPD soon enough before surgery.\n\nIt was challenging to identify patients with COPD at surgical clinics and multidisciplinary meetings as patients had just received a diagnosis of cancer, but a definitive treatment plan had yet to be instituted. At this point the focus is on the surgical condition rather than medical conditions such as COPD. Medical records focused mostly upon surgical condition and respiratory records were often in separate (unavailable) notes and smoking histories were rarely present. This made screening laborious and time inefficient.\n\nThe definitive decision on whether to operate would only be made following neoadjuvant treatment. Oncology clinics were assessed as an identification point, but we found that potential participants attended too many different clinics to find a suitable point for screening.\n\nScreening the electronic patient record for patients scheduled for surgery did not successfully identify additional people with COPD. Therefore, COPD was often not formally diagnosed until the following the anaesthetic preoperative assessment clinic which usually occurred 2–3 weeks before surgery, with cardiopulmonary exercise testing (CPET) testing taking place at a similar time before surgery.\n\nThe difficulty in identifying potential participants with COPD was somewhat unexpected. As audit data from the pre-operative cardiopulmonary exercise testing clinics in both Oxford and Newcastle-upon-Tyne suggested that COPD was present in 10 to 15% of the 2,000 to 3,000 patients each year passing through those clinics. This meant that our pool of potential participants was around 300 in each centre each year.\n\nVascular surgery clinics were also assessed; these non-cancer patients have a more clearly defined pre-surgical pathway. However, we found that due to changes in surgical practices, most patients with respiratory disease were treated endovascularly and thus recruiting from this clinic was also deemed low yield.\n\nAt anaesthetic pre-assessment clinics, the main challenge was that potential participants with undiagnosed COPD may not have been formally diagnosed after the pre-assessment clinic (when patients were sent for lung function tests); this made confirmation of eligibility difficult, and further lessened time for study inclusion.\n\nIn Newcastle upon Tyne, surgical patients attend the anaesthetic pre-assessment clinic about one month before surgery, this is in contrast to Oxford where the time between anaesthetic assessment and surgery is often much shorter. In Newcastle-upon-Tyne we were more successful at identifying patients with COPD, but despite this only two patients were recruited into the study (one of whom subsequently withdrew).\n\nWe discussed increasing the number of sites for the study with three other potential UK sites (two teaching hospitals and one large district general hospital) who performed evaluations of their services, taking into account the preliminary findings of this work. This would help us evaluate whether the identification and recruitment issues were generalisable to other centres. However, we found that in all three centres the main point of identifying COPD was found to be at anaesthetic pre-assessment clinics, which occur two to three weeks prior to operation date (similar to Oxford).\n\nCPS, in his role as Royal College of Anaesthetists National Clinical Lead for Perioperative Medicine, surveyed perioperative medicine and preoperative assessment clinics in 110 hospitals in England over the course of 201728. This piece of work found that the usual time interval between anaesthetic preassessment and surgery was often only 2–3 weeks, but with wide variability (unpublished observations). This is equivalent to current practice in Oxford.\n\n\nDiscussion\n\nPulmonary rehabilitation is a potentially valuable treatment for improving the health status of people with COPD prior to surgery. We established that a full randomised controlled trial is not feasible. As a result of this study we have identified an unmet need in the early identification of COPD in patients presenting for surgery.\n\nAlthough the study was only run in two UK centres, further scoping work in three additional centres and a related England-wide survey of anaesthetic services means that we are reasonably confident that similar challenges in identification and recruitment would be found if a randomised controlled trial were to run across the UK, and thus we believe that our findings are generalisable.\n\nPulmonary rehabilitation is an integral part of the NICE guidelines for the treatment of COPD, and therefore every person with COPD should be offered this treatment (alongside the other components of therapy recommended by NICE). This raises the question about whether a randomised controlled trial is actually the most appropriate methodology for future work. Barriers to pulmonary rehabilitation are well recognised, even when implemented as a clinical treatment15,16. These barriers can be even more pronounced when tested as an optional research intervention29. We provided free transport and offered flexible scheduling for potential participants. These were recommended by our patient liaison group during the study design phase to help overcome barriers to taking part in pulmonary rehabilitation, but clearly were insufficient to enable us to recruit at a sufficient rate.\n\nWe therefore speculate that if it we could identify COPD at the beginning of the patient's surgical journey, patients would be much better placed to have appropriate management and optimisation of their COPD. Spirometry is cheap, widely available and reliable; and thus perfect for a simple primary care test which should be offered much more widely, and would allow for early optimisation of drug therapy. Pulmonary rehabilitation could occur in a more timely fashion as this intervention could be integrated and planned alongside chemo- and radio- therapy, rather than in the weeks immediately preceding surgery. Patients with COPD would benefit even if they do not eventually proceed to surgery, with potential cost savings for the NHS14,30. Potential solutions to this are illustrated in Figure 3.\n\nThis study has demonstrated the considerable challenge in performing additional interventions in the immediate period before surgery. However, the duration of the patient’s journey from referral to surgery can take several months and remains an ideal period to optimise COPD if appropriate patients are identified earlier in the process. Some UK hospitals have recently implemented initiatives to ‘re-design’ this surgical pathway, which may help overcome this barrier31. The importance of identifying and engaging with patients early after the “moment of contemplation” of surgery is clearly a critical success factor for interventions such as pulmonary rehabilitation; which are known (from other contexts) to require a defined period of time to implement and provide benefit. However, we should take caution from evidence from studies in lung cancer which show that the time of diagnosis is a difficult time to consider pulmonary rehabilitation32. Although there may be an opportunity to provide pulmonary rehabilitation whilst neo-adjuvant therapy is being provided patients often do not want to engage with pulmonary rehabilitation at a time when they are dealing with a new, life changing diagnosis and having burdensome, potentially toxic cancer treatment14. Thus, it might turn out that pulmonary rehabilitation can only really feasibly delivered once cancer treatment has finished. There is emerging evidence that exercise therapies enhance cancer survival33,34 and that a recommendation from the oncologist may be influential in the view patients might take.\n\nPulmonary rehabilitation represents an important part of the NICE guidelines for the treatment of COPD and is readily available in the NHS. We know that patients will benefit from pulmonary rehabilitation, even if it is consequently shown not to have a specific effect upon postoperative pulmonary complications. Patients who consequently do not require surgery will still benefit. We have shown that a randomised controlled trial is not feasible, so we need to approach this in a different way using alternate methodologies.\n\nEthical approval was granted by the South Yorkshire Research Ethics Committee (approval number 17/YH/0220). Written informed consent was obtained from all participants prior to the start of the study.\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: CONSORT checklist and flow diagram for ‘Findings of a feasibility study of pre-operative pulmonary rehabilitation to reduce post-operative pulmonary complications in people with chronic obstructive pulmonary disease scheduled for major abdominal surgery’. https://doi.org/10.17605/OSF.IO/4ZQNK35\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nBrock A, Griffiths C, Rooney C: The impact of introducing ICD-10 on analysis of respiratory mortality trends in England and Wales. Health Stat Q. 2006; (29): 9–17. PubMed Abstract\n\nNICE: Chronic obstructive pulmonary disease quality standard [QS10]. 2011. Reference Source\n\nFernandez-Bustamante A, Frendl G, Sprung J, et al.: Postoperative Pulmonary Complications, Early Mortality, and Hospital Stay Following Noncardiothoracic Surgery: A Multicenter Study by the Perioperative Research Network Investigators. JAMA Surg. 2017; 152(2): 157–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta H, Ramanan B, Gupta PK, et al.: Impact of COPD on postoperative outcomes: results from a national database. Chest. 2013; 143(6): 1599–606. PubMed Abstract | Publisher Full Text\n\nFields AC, Divino CM: Surgical outcomes in patients with chronic obstructive pulmonary disease undergoing abdominal operations: An analysis of 331,425 patients. Surgery. 2016; 159(4): 1210–6. PubMed Abstract | Publisher Full Text\n\nPearse RM, Moreno RP, Bauer P, et al.: Mortality after surgery in Europe: a 7 day cohort study. Lancet. 2012; 380(9847): 1059–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nA National Five Year Plan for Lung Health. Reference Source\n\nMcCarthy B, Casey D, Devane D, et al.: Pulmonary rehabilitation for chronic obstructive pulmonary disease. Cochrane Database Syst Rev. 2015; (2): CD003793. PubMed Abstract | Publisher Full Text\n\nSpruit MA, Singh SJ, Garvey C, et al.: An official American Thoracic Society/European Respiratory Society statement: key concepts and advances in pulmonary rehabilitation. Am J Respir Crit Care Med. 2013; 188(8): e13–64. PubMed Abstract | Publisher Full Text\n\nSeymour JM, Moore L, Jolley CJ, et al.: Outpatient pulmonary rehabilitation following acute exacerbations of COPD. Thorax. 2010; 65(5): 423–8. PubMed Abstract | Publisher Full Text\n\nPuhan MA, Scharplatz M, Troosters T, et al.: Respiratory rehabilitation after acute exacerbation of COPD may reduce risk for readmission and mortality -- a systematic review. Respir Res. 2005; 6: 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPuhan MA, Gimeno-Santos E, Scharplatz M, et al.: Pulmonary rehabilitation following exacerbations of chronic obstructive pulmonary disease. Cochrane Database Syst Rev. 2011; (10): CD005305. PubMed Abstract | Publisher Full Text\n\nMan WD, Polkey MI, Donaldson N, et al.: Community pulmonary rehabilitation after hospitalisation for acute exacerbations of chronic obstructive pulmonary disease: randomised controlled study. BMJ. 2004; 329(7476): 1209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffiths TL, Phillips CJ, Davies S, et al.: Cost effectiveness of an outpatient multidisciplinary pulmonary rehabilitation programme. Thorax. 2001; 56(10): 779–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayton C, Clark A, Olive S, et al.: Barriers to pulmonary rehabilitation: characteristics that predict patient attendance and adherence. Respir Med. 2013; 107(3): 401–7. PubMed Abstract | Publisher Full Text\n\nMcCarron EP, Bailey M, Leonard B, et al.: Improving the uptake: Barriers and facilitators to pulmonary rehabilitation. Clin Respir J. 2019; 13(10): 624–629. PubMed Abstract | Publisher Full Text\n\nNICE: NIfHaCE. Chronic obstructive pulmonary disease in over 16s: diagnosis and management NICE guideline [NG115]. 2019. Reference Source\n\nRoyal College of Physicians: National COPD Audit Programme - Pulmonary rehabilitation: Time to breathe better. 2015. Reference Source\n\nYamana I, Takeno S, Hashimoto T, et al.: Randomized Controlled Study to Evaluate the Efficacy of a Preoperative Respiratory Rehabilitation Program to Prevent Postoperative Pulmonary Complications after Esophagectomy. Dig Surg. 2015; 32(5): 331–7. PubMed Abstract | Publisher Full Text\n\nShakouri SK, Salekzamani Y, Taghizadieh A, et al.: Effect of respiratory rehabilitation before open cardiac surgery on respiratory function: a randomized clinical trial. J Cardiovasc Thorac Res. 2015; 7(1): 13–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMujovic N, Mujovic N, Subotic D, et al.: Preoperative pulmonary rehabilitation in patients with non-small cell lung cancer and chronic obstructive pulmonary disease. Arch Med Sci. 2014; 10(1): 68–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenzo R, Wigle D, Novotny P, et al.: Preoperative pulmonary rehabilitation before lung cancer resection: results from two randomized studies. Lung Cancer. 2011; 74(3): 441–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaeda K, Higashimoto Y, Honda N, et al.: Effect of a postoperative outpatient pulmonary rehabilitation program on physical activity in patients who underwent pulmonary resection for lung cancer. Geriatr Gerontol Int. 2016; 16(5): 550–5. PubMed Abstract | Publisher Full Text\n\nFishman A, Martinez F, Naunheim K, et al.: A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. N Engl J Med. 2003; 348(21): 2059–73. PubMed Abstract | Publisher Full Text\n\nRies AL, Make BJ, Lee SM, et al.: The effects of pulmonary rehabilitation in the national emphysema treatment trial. Chest. 2005; 128(6): 3799–809. PubMed Abstract | Publisher Full Text\n\nSewell L, Singh SJ, Williams JE, et al.: How long should outpatient pulmonary rehabilitation be? A randomised controlled trial of 4 weeks versus 7 weeks. Thorax. 2006; 61(9): 767–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBolton CE, Bevan-Smith EF, Blakey JD, et al.: British Thoracic Society guideline on pulmonary rehabilitation in adults. Thorax. 2013; 68 Suppl 2): ii1–30. PubMed Abstract | Publisher Full Text\n\nBougeard AM, Brent A, Swart M, et al.: A survey of UK peri-operative medicine: pre-operative care. Anaesthesia. 2017; 72(8): 1010–5. PubMed Abstract | Publisher Full Text\n\nKochovska S, Fazekas B, Hensley M, et al.: A Randomized, Double-Blind, Multisite, Pilot, Placebo-Controlled Trial of Regular, Low-Dose Morphine on Outcomes of Pulmonary Rehabilitation in COPD. J Pain Symptom Manage. 2019; 58(5): e7–e9. PubMed Abstract | Publisher Full Text\n\nBurns DK, Wilson EC, Browne P, et al.: The Cost Effectiveness of Maintenance Schedules Following Pulmonary Rehabilitation in Patients with Chronic Obstructive Pulmonary Disease: An Economic Evaluation Alongside a Randomised Controlled Trial. Appl Health Econ Health Policy. 2016; 14(1): 105–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrocott MPW, Plumb JOM, Edwards M, et al.: Re-designing the pathway to surgery: better care and added value. Perioper Med (Lond). 2017; 6: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteiner MC: Should pulmonary rehabilitation be a standard of care in lung cancer? Thorax. 2019; 74(8): 725–6. PubMed Abstract | Publisher Full Text\n\nCormie P, Zopf EM, Zhang X, et al.: The Impact of Exercise on Cancer Mortality, Recurrence, and Treatment-Related Adverse Effects. Epidemiol Rev. 2017; 39(1): 71–92. PubMed Abstract | Publisher Full Text\n\nCOSo A: COSA position statement on exercise in cancer care. 2018. Reference Source\n\nMarlow L: Findings of a feasibility study of pre-operative pulmonary rehabilitation to reduce post-operative pulmonary complications in people with chronic obstructive pulmonary disease scheduled for major abdominal surgery. 2020. http://www.doi.org/10.17605/OSF.IO/4ZQNK"
}
|
[
{
"id": "71179",
"date": "30 Sep 2020",
"name": "Ianthe Boden",
"expertise": [
"Reviewer Expertise Physiotherapy",
"critical care",
"perioperative medicine",
"health economics",
"clinical trials",
"postsurgical recovery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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-conceived and beautifully reported feasibility study that provides vital information required by clinical trialists and grant funding bodies to consider the design of future research in the field of pre-operative optimisation in this high-risk surgical cohort.\nA preexisting diagnosis of COPD significantly increases a person's risk of suffering a complication after major surgery. There is preliminary evidence that for patients with COPD awaiting thoracic surgery that pulmonary rehabilitation (a supervised twice-weekly program of tailored education and physical exercise) in the weeks immediately prior to surgery may reduce the risk of postoperative complications and improve recovery after surgery. Pulmonary rehabilitation is an established clinical program within developed countries. There is definitive evidence of the benefit and cost-effectiveness of pulmonary rehabilitation as a primary therapy to improve symptoms and quality of life for people with stable COPD. However, it is yet to be established as an effective program to preoptimise patients with COPD awaiting major abdominal surgery. This is an intriguing possibility.\nThis paper reports the results of a multicentre randomised controlled trial that assessed the feasibility of conducting a trial of preoperative pulmonary rehabilitation for patients with COPD and awaiting major abdominal surgery. This trial was pre-registered with feasibility outcomes specified a priori.\nDespite a strong research group with significant experience and track record, two well-resourced centers of excellence as investigating sites, and a well concieved protocol to provide evidence based preoperative pulmonary rehabilitation, this trial was found to be unfeasible due to exceptionally low recruitment uptake.\nDespite all their best efforts (free transport to pulmonary rehabilitation and flexible appointment times) the reasons were reported as being predominately outside the control of researchers; namely established presurgical pathways and finite time scale from time of listing for surgery to date of surgery often being shorter (2 - 3 weeks) than the time period allowable of being able to provide an effective dose of preoperative pulmonary rehabilitation (4 - 6 weeks).\nHowever, I was unable to determine the exact proportions of the various reasons for eligible patients to not be recruited into the trial as the numbers in the flow chart in the box of 'Declined' patients do not add up to the total stated of n=42. I would also suggest that the 'not approached' group are actually ineligible/not suitable as they are not listed for surgery so no longer are eligible criteria. The flow chart's numbers are all a bit off with boxes following the arrows of flow not adding up to the box prior to it in flow order. This requires clarification.\nA few other comments that could be considered within reporting of this paper:\nIt was unclear how a diagnosis of COPD was determined in the eligibility criteria. GOLD standard? Spirometry? Medical record report/coding?\n\nThe discussion considers that to overcome the barriers identified that the pre-surgical pathway would need to be altered to ensure that a COPD morbidity is recognised earlier and these patients are fed into a pulmonary rehab program at a much earlier time frame than the preoperative anaesthetic assessment clinic. I would be interested to hear from the authors what interventions have been recently tested within the CURRENT surgical pathways that prevent postoperative complications e.g preoperative physiotherapy to teach patients breathing exercises inserted into preanaesthetic clinics (Boden et al.) or inspiratory muscle training (Kendall et al.), and how these could be considered within this specific surgical cohort of patients with COPD. Should more be done to implement these findings within existing pathways? Or should energy be spent in changing the pathway to get more patients into prehab/pulmonary rehabilitation?\n\nAlso, what reflections do the authors have of their findings in contrast to the emerging body of evidence for 'pre-habilitation' in a general surgical cohort (not refined to COPD)? Recent Spanish (Barabaran Garcia et al.) and Canadian (Carli et al.) studies did not appear to have limitations to recruiting to a high-intensity preoperative exercise program within 4-6 weeks of surgery. Is this a factor related to the cohort of patients with COPD or related to the different countries? Or something else? A UK trial of prehab for vascular surgery patients also did not have issues with recruitment. I think it would be wise for the authors to discuss these differences in trial conduct.\n\nThe authors should be congratulated on ensuring that this data is available and published in the interests of transparent reporting. It is often disheartening to report null results or trial conduct 'failures'. However, in the interests of future efficient research activities, the paper published here presents excellent information and thought-provoking concepts that challenge our thinking about how, when, and what to do with patients prior to surgery to improve their postoperative outcome. Thankfully the authors did not bury their data along with their disappointment when their hypothesis was unable to be tested to its fullest. This paper is an excellent point of learning for all of us working in the perioperative medicine sector. Thank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "61114",
"date": "26 Nov 2020",
"name": "Gary Minto",
"expertise": [
"Reviewer Expertise perioperative 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\nFeasibility of an RCT of pulmonary rehab for 3 weeks vs standard of care in COPD patients before major surgery.\nReally well written - clear statements of the scale of the problem and clear statements of the need for evidence. Clear rationale for plausibility of intervention. Clear focus on the purpose of THIS study - is pre-operative pulmonary rehabilitation feasible? (rather than does it work).\nDecision-making points about whether to proceed to a “full” study were defined and the feasibility endpoints were well designed.\nEven the methodology BEFORE feasibility acknowledges a likely problem - authors were assuming a drop out of 1/3 of pts who had been recruited! - “this 48 dataset target, based on known drop-out rates from pulmonary rehabilitation and potential further data loss due to surgical scheduling, we anticipated that we would need to recruit 72 patients”.\nPoints of the pathway to identify potentially eligible participants comprehensively explored - Only 3 randomised! Thorough evaluation of why - i.e. issues identified related to the confirmation of diagnosis of COPD within a pathway towards major body cavity surgery. Not only in the 2 participating centres but also in 3 further centres (in detail) plus findings from a UK national survey of preop units.\nIMPLICATIONS for future research: Agree wholeheartedly with the authors' conclusion that under current UK surgical pathway conditions, an RCT of pulmonary rehab as prehabilitation for COPD before major surgery is not feasible. There is a commendable readiness on the part of the investigators to change their outlook in the face of this feasibility study evidence.\nWhy is it that such a large proportion of the patients who WERE identified as having COPD and were eligible (n = 45 , figure 2), declined to participate - often on the basis that they had “too many appointments/too much to think about/too far to travel? As the authors put it: “patients often do not want to engage with pulmonary rehabilitation at a time when they are dealing with a new, life changing diagnosis and having burdensome, potentially toxic cancer treatment\".\nThe authors make a key point that when pulmonary rehab is framed as an “optional” research extra, then it seems acceptable to opt out. I concur with their suggestion that pulmonary rehab could instead be framed (in those hospitals which have the resource) as an important part of the surgical pathway; and that future research into effectiveness might be better conducted by comparing clinical outcomes after surgery in hospitals with or without “Surgery School” (in a manner akin to research into the effectiveness of Enhanced Recovery Pathways).\nIMPLICATIONS for practice: It is striking that pulmonary rehabilitation is a proven and effective therapy (with a great benefit in terms of QALYs) which is meant to be offered to ALL patients with a diagnosis of COPD, yet so few patients with COPD (300 participants per year being assessed for major surgery in each of the 2 sites are estimate to have the condition) arrived at the preop clinic having already had experience of pulmonary rehab. So wider questions - implied by the authors in the discussion, but perhaps worth stating explicitly are things like:\nWhy are so many people with COPD undiagnosed? Why do many of those with a diagnosis still have no personal experience of pulmonary rehabilitation? Why are we having to use attendance at hospital for assessment for surgery as an opportunistic moment to introduce pulmonary rehabilitation?\nThese might be addressed by better education of the general population and health care professionals.\n\nA philosophical question: Is it ethical to proceed to perform major surgery when patients have opted out of engaging with prehabilitation? In the current scenario there is (very) limited clear cut evidence that pulmonary rehabilitation has utility and might improve clinical outcomes after surgery? It appears that an RCT of this therapy isn't possible in the UK without a radical overhaul of surgical pathways. How should we now proceed to find this evidence (whichever way the evidence pans out)?\nMinor issues: Please make sure the pulmonary rehab program is clearly defined. In the introduction - pulmonary rehab is explained as 1 hour of exercise and 1 hour of education twice a week for 6 weeks. - What is the format of education delivered for 2 hours a week for 6 weeks?\nAttendance - the logistics of getting to classes appears to be a problem or possibly a factor in refusals. Could classes be virtual?\nFigure 2 - typo in box 3 left hand side - should read 45 were approached (not 65). Page 4 top line - is this a typo? exclusions: “Patients scheduled cardiac, thoracic and orthopaedic surgery and orthopaedic surgery\" Page 8 typo? \"COPD may not have been formally diagnosed (UNTIL) after the pre-assessment clinic\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": []
}
] | 1
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https://f1000research.com/articles/9-172
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https://f1000research.com/articles/9-121/v2
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09 Mar 20
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{
"type": "Research Article",
"title": "Genomic and protein structure modelling analysis depicts the origin and pathogenicity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China",
"authors": [
"Ning Dong",
"Xuemei Yang",
"Lianwei Ye",
"Kaichao Chen",
"Edward Wai-Chi Chan",
"Sheng Chen",
"Ning Dong",
"Xuemei Yang",
"Lianwei Ye",
"Kaichao Chen",
"Edward Wai-Chi Chan"
],
"abstract": "Background: A pandemic outbreak caused by a novel coronavirus, 2019-nCoV, has originated from Wuhan, China and spread to many countries around the world. The outbreak has led to around 45 thousand cases and over one thousand death so far. Methods: Phylogenetic analysis and sequence alignment were used to align the whole genome sequence of 2019-nCoV with other over 200 sequences of coronaviruses to predict the origin of this novel virus. In addition, protein modeling and analysis were performed to access the potential binding of the spike protein of 2019-nCoV with human cell receptor, angiotensin-converting enzyme 2 (ACE2). Results: Detailed genomic and structure-based analysis of a new coronavirus, namely 2019-nCoV, showed that the new virus is a new type of bat coronavirus and is genetically fairly distant from the human SARS coronavirus. Structure analysis of the spike (S) protein of this new virus showed that its S protein only binds much weaker to the ACE2 receptor on human cells whereas the human SARS coronavirus exhibits strongly affinity to the ACE receptor. Conclusions: These findings suggest that the new virus should theoretically not be able to cause very serious human infection when compared to human SARS virus. However, the lower pathogenicity of this new virus may lead to longer incubation time and better adaption to human, which may favor its efficient transmission in human. These data are important to guide design of infection control policy and inform the public on the nature of threat imposed by 2019-nCov. Most importantly, using the analysis platform that we have developed, we should be able to predict whether the new mutations could lead to the increase of infectivity of the mutated virus in a very short time.",
"keywords": [
"2019-nCoV",
"Genomics",
"Protein modelling",
"Origin",
"Pathogenicity",
"Wuhan"
],
"content": "Introduction\n\nA cluster of pneumonia cases of unknown cause were reported in Wuhan, the capital City of Hubei Province of China in December 2019. On 18th January 2020, a total of 44 such cases were documented, among whom two patients have died, five in critical condition, and six have been discharged from hospital. Most patients had visited or worked in a seafood wholesale market in Wuhan. In the early stage of this outbreak, there is no strong evidence which suggests that the unknown agent is highly infectious as no health care personnel in the hospitals where the patients were admitted were infected. However, the situation changed dramatically with newly infected cases rapidly increased and human to human transmission has been confirmed with several health care personnel being confirmed to be infected. As of Jan 28, 2020, the infection cases sharply increased in the past few days reaching a total of 4500 cases with over 100 confirmed deaths. Sporadic new cases were reported in more and more provinces in China mainly due to the traveler from Wuhan. Many confirmed cases were reported in many other countries. These patients were known to originated from or have been to Wuhan. These epidemiological data were quite different from the data reported in the beginning and may suggest that the new virus could undergo human host adaption/evolution and become more adaptive to human host leading to more efficient human to human transmission.\n\nAs of February 11, 2020, a total of 42,714 confirmed infections and 21,675 suspected cases were reported worldwide with over 31,728 confirmed cases in Hubei Province according to data from the National Health Commission of China. A total of 1,017 deaths were reported so far with 974 deaths being from Hubei Province. The mortality rate is very high within Hubei Province reaching 3.06% (974/31,728), while the mortality rate outside Hubei Province was about 0.39% (43/10986). These data suggested that the 2019-nCoV is very infectious, while the pathogenicity seemed to be lower than SARS virus. It is urgent to find scientific evidence to support the epidemiological data to guide further control measure development.\n\n\nMethods\n\nA total of 211 genome sequences of viruses in the Coronaviridae family including the 2019-nCoV Wuhan virus were downloaded from GenBank (last accessed on 11 January 2020; list of downloaded sequences is provided as extended data22). Circular proteomic trees were computed using ViPTreeGen v1.1.21. The sequence of a Breda virus (accession: NC_007447) was used as an outgroup. Alignment of sequences in different viral genomes was conducted using the alignment function of ViPTree1. The phylogenetic tree and sequence alignment products were manually edited using Inkscape v0.912. Ten spike protein sequences which were similar to that of 2019-nCoV were downloaded from NCBI. SNP analysis was performed using Mega X3, and the alignment was carried out by using ClustalW4. The aligned sequences were edited and viewed in CLC Genomics Workbench 20.\n\nSpike receptor-binding domain (RBD) of coronavirus 2019-nCoV and four bat-originated coronavirus were predicted by aligning their spike protein sequences to spike RBD of SARS coronavirus5 using Clustal Omega6. Homology modeling of spike proteins and the related RBDs of 2019-nCoV and four bat-originated coronavirus was performed using SARS-CoV spike glycoprotein as template (PDB ID: 5X58)7 on the Swiss-Model workspace8. The structure assessment results are presented below. The models were visualized with PyMol. The contacts between human ACE2 and spike RBD were predicted by aligning to structure of the RBD in complex with the human receptor ACE2 (PDB ID: 2AJF)5.\n\nSequence alignment performed with CLC Genomics Workbench can be replicated using open source alternatives such as ClustalW.\n\nAn open source version of PyMol has been made freely available by the developers, available from GitHub (https://github.com/schrodinger/pymol-open-source).\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/2020.01.20.913368).\n\n\nResults and discussion\n\nOn 6th January 2020, the Chinese authority released the sequence (accession#: MN908947) of a novel coronavirus, designated as 2019-nCoV, which was isolated from one of the pneumonia patients and confirmed to be the causative agent for this outbreak9. Coronaviruses are a large family of viruses, most of which cause mild infections such as the common cold, but some such as the SARS and MERS (Middle East Respiratory Syndrome) viruses cause severe and potential fatal respiratory tract infections10,11. Some coronaviruses are known to be transmitted easily between humans, while others do not. Based on currently available information, the 2019-nCoV virus belongs to a category that can cause severe illness in some patients but does not transmit readily between people12–14. It is necessary to investigate the genetic and functional data of this new virus and compare to other coronaviruses so as to guide future research and design of appropriate infection control policy to prevent widespread dissemination of another potentially deadly coronavirus since the emergence of the SARS and MERS viruses. In this study, we performed in-depth genetic analysis of 2019-nCoV and generated data which provide timely and valuable insight into the potential origin of this virus, its ability to cause human infection, and its genetic relatedness with SARS and MERS.\n\nPhylogenetic analysis of genomic sequences of coronaviruses deposited in the GenBank revealed that 2019-nCoV belonged to betacoronavirus and exhibited the closest linkage with two SARS-like coronavirus from bat (bat-SL-CoVZX45 and bat-SL-CoVZX21) (Figure 1a). According to the phylogenetic tree, human SARS viruses were closest to bat SARS-like viruses but with a lesser degree to bat coronaviruses, and was least related to other coronaviruses. The 2019-nCoV stands in a position between bat SARS-like viruses and bat coronaviruses, suggesting it is less related to the human SARS virus than other bat SARS-like viruses, and is likely a new type of bat coronavirus. Nevertheless, all coronaviruses that exhibit close linkage with 2019-nCoV originated from bat, strongly suggesting that this new coronavirus originated from bat (Figure 1a). Coronaviruses of other species including the murine coronavirus are genetically distant from this new coronavirus, indicating that 2019-nCoV did not originate from other animal hosts. As bats are not sold in the Wuhan market, animals that serve as the transmission vehicle remains to be identified15,16.\n\n(a) Phylogenetic tree of coronaviruses from different species. The type of coronovirus and the host were labelled. Virus labeled with red is the newly discovered coronovirus 2019-nCoV. (b) Sequence alignment of representative the new 2019-nCoV, bat SARS like coronoviruses and bat coronoviruses. These included two highly homologous human SARS coronaviruses: SARS CoV P2 (FJ882963) and SARS CoV ZJ02 (EU371559), one bat SARS virus showing high homology with human SARS virus and similar potential to infect human as human SARS coronavirus: bat SARS CoV W1V1 (KF367457), two bat SARS-like viruses that are not able to infect human: (bat-SL-CoVZX45 and Rp Shaanxi2011) and the newly discovered 2019-nCoV from Wuhan.\n\nThe sequence of 2019-nCoV was annotated and aligned with several representative coronaviruses selected according to the degree of genetic relatedness depicted by the phylogenetic tree (Figure 1b). These included two highly homologous human SARS coronavirus: SARS CoV P2 (FJ882963) and SARS CoV ZJ02 (EU371559), one bat SARS virus that exhibits high homology with human SARS virus and similar potential to infect human as human SARS coronavirus: bat SARS CoV W1V1 (KF367457)17, two bat SARS-like viruses that were not able to infect human: (bat-SL-CoVZX45 and Rp Shaanxi2011), and two un-related coronaviruses, the MERS virus MERS CoV (NC019843) and the Avian Infectious Bronchitis (IBV) virus IBV CoV (AY646283). The new 2019-nCoV was annotated slightly different from the human SARS virus and other coronaviruses, but the functionally important ORFs, ORF1a and ORF1b, and major structural proteins including the spike (S), membrane (M) and envelop (E) and nucleic capsid (N) proteins are well annotated (Figure 1b). Consistent with the phylogenetic tree data, 2019-nCoV did not align well with the MERS and IBV virus (Figure S1, Extended data22). Among the SARS viruses, bat coronaviruses and 2019-nCoV, the non-structural proteins generally aligned well but variations were observable in major structural proteins and some small ORFs (Figure 1b). Detailed sequence alignment showed that 2019-nCoV exhibited significant sequence variation at several regions with the human SARS coronavirus including the N-terminal region of ORF1a and S protein, ORF3, E, ORF6, 7 and 8, and the middle part of N protein. Bat SARS-like virus W1V1 aligned well with human SARS virus P2, with some variations at ORF8 and an insertion between ORF6 and 7. Coronavirus 2019-nCoV aligned best with the bat SARS-like virus bat-SL-CoVZX45, with the majority of genetic variations being seen at the N-terminal part of S protein. The bat-SL-CoVZX45 virus itself exhibited a high degree of variation with another bat SARS-like CoV Rp Shaanxi2011 at ORF1a, the N-terminus of S protein and other structural proteins. However, bat SARS-like CoV Rp Shaanxi2011 exhibited high homology with human SARS virus ZJ02, with variation being seen at the N-terminus of S protein and the middle part of N protein (Figure 1b). To check if 2019-nCoV is more close to bat SARS like virus or bat coronavirus, we selected two adjacent viruses from each group, bat SARSCoV Rf1/2004 and bat coronavirus BM48-31/BGR/2008, to perform alignment with the new virus with result showing that 2019-nCoV showed similar diversity from these two strains (Figure S1b, Extended data22). When 2019-nCoV was aligned with bat CoV HKU9-1, another bat coronavirus with further distance, it showed that 2019-nCoV was very different from this virus (Figure S1c, Extended data22). These sequence alignment data were consistent with results of phylogenetic tree analysis and indicated that 2019-nCoV exhibited in between bat SARS like viruses and bat coronaviruses, but is genetically distant from the human SARS virus. It should be considered a new type of bat coronavirus.\n\nSince the S protein is the protein that exhibits the highest degree of genetic variations among different coronaviruses, we performed phylogenetic analysis of the S protein of different coronaviruses (Figure S2, Extended data22). Our data showed that the S protein of 2019-nCoV exhibited high homology with bat SARS-like coronaviruses such as bat-SL-CoVZX45 and bat-SL-CoVZX21, human SARS virus and bat coronaviruses. Homology of S protein of 2019-nCoV with representative human SARS virus, bat SARS like viruses and bat coronaviruses was determined as shown in Table 2. S protein of 2019-nCoV showed about 76% homology to human SARS virus P2 and high homology to bat SARS like viruses, while it showed 72% homology to closest bat coronavirus, bat coronavirus BM48-31, even lower homology with other bat coronaviruses. These data further suggested that 2019-nCoV is more likely a new type of bat coronavirus with only loose linkage with the SARS virus. Interestingly, different regions of the S proteins exhibited different levels of homology among the known coronaviruses (Figure 1b). Amino acid sequence alignment showed consistently that the N-terminal regions were far more diverse than the C-terminus, which seemed to be highly conservative (Figure 2). Aligning these regions to the structure of S protein indicated that the structurally conserved C-terminal aligned well to the transmembrane domain which consists of a double helix, whereas the most variable region aligned to the N-terminal domain; on the other hand, the receptor binding domain exhibited intermediate level of sequence variation (Figure 2).\n\nUpper panel is the amino acid sequence alignment and the lower panel is the structure of the S protein. Three domains of S protein were labelled as red, blue and green, which aligned with the different colors for the aligned sequence. Sequences that exhibits the highest degree of variation are in the N-terminal domain, followed by those in the RBD. The C-terminal green domain is the most conservative among the test viruses.\n\nDue to the high amino acid sequence homology of the S protein in 2019-nCoV and the SARS virus which can cause severe human infection, we analyzed the structural similarity of this protein in various viruses. Protein structure modeling was performed to obtain high quality structure of S proteins from different coronaviruses (Table 1). The high level similarity observable between the structures of S protein from different viruses implied that the S protein of 2019-nCoV and bat coronaviruses would most likely use the same human cell receptor as SARS virus (Table 2). It was shown that angiotensin-converting enzyme 2 (ACE2) was the cellular receptor of the SARS virus S protein18. Complex structure of ACE2 with the receptor binding domain (RBD) of S protein of SARS virus has been resolved and demonstrated tight interaction between these two proteins at the interaction interface5. Current studies have confirmed that 2019-nCoV indeed showed binding to ACE2. These data prompted us to determine the level of interaction between the S protein of 2019-nCoV with its potential cellular receptor ACE2. Using modeled S protein structure and by further performing structure-based alignment, we obtained the complex structure of RBD of the S protein of 2019-nCoV and several bat coronaviruses with human ACE2, using the complex structure of RBS of S protein from human SARS virus and ACE2 (2ajf) as reference5. Structural analysis of potential interactions between RBD of S protein from human SARS virus and ACE2 protein depicted several interaction points including four hydrophobic interactions: ACE2(Y41)/RBD(Y484), ACE2(L45)/RBD(Y484), ACE2(L79, M82)/RBD(L472), ACE2(Y83)/RBD(Y475), one salt-bridge: ACE2(E329)/RBD(R426) and one cation-π interaction: ACE2(K353)/RBD(Y491) (Figure 3a–e). However, examination of interaction between RBD of 2019-nCoV and human ACE2 depicted only one potential hydrophobic interaction between ACE2(L79, M82) and RBD(F486) and one cation-π interaction ACE2(K353)/RBD(Y492). Further examination of interactions between RBD from bat SARS like coronaviruses, bat-SL-CoVZX45 and bat coronavirus HKU3-1 that do not infect human, showed only one cation-π interaction interaction, ACE2(K353)/RBD(Y481)20,21. Another bat-originated coronavirus, bat SARS CoV W1V1 that displayed strong binding to ACE2 and exhibited potential to cause human infection was also included for analysis17. Binding affinity of RBD from this virus to ACE2 was as tight as that of the human SARS virus, involving four hydrophobic interactions: ACE2(Y41)/RBD(Y485), ACE2(L45)/RBD(Y485), ACE2(L79, M82)/RBD(F473), ACE2(Y83)/RBD(Y476), one salt-bridge: ACE2(E329)/RBD(R427) and one cation-π interaction site ACE2(K353)/RBD(Y492). These data suggested that the higher binding affinity of RBD of coronavirus to ACE2 will confer the virus higher infectivity and pathogenicity. The fact that the RBD of 2019-nCoV exhibited much lower affinity to ACE2 implies that the virulence potential of 2019-nCoV should be much lower than that of human SARS virus, but is nevertheless stronger than viruses that do not cause human infection20,21; such finding is also consistent with the current epidemiological datain that 2019-nCoV only caused severe pneumonia in patients with weaker immune system such as the elderlies and people with underlying diseases. The weaker binding affinity of 2019-nCoV to human cell might also explain the limited human to human transmission potential of this virus observed to date.\n\n1, Structure assessment. MolProbity is all-atom contact analysis based only on properties of the predicted model. Lower numbers indicate better models. Ramachandran favoured indicates energetically favoured regions for backbone dihedral angles against amino acid residues in protein structure. Larger numbers indicate better models.\n\n2, Model evaluation. GMQE (Global Model Quality Estimation) is a quality estimation which combines properties of the target–template alignment and the template search method. The resulting GMQE score is expressed as a number between 0 and 1, larger numbers indicate higher reliability. The QMEAN Z-score provides an estimate of the \"degree of nativeness\" of the structural features observed in the model on a global scale. QMEAN Z-scores around zero indicate good agreement between the model structure and experimental structures of similar size.\n\nInteractions between ACE2 with RBD of human SARS virus (a), highly similar bat SARS like coronavirus, CoV-W1V1 that can infect human (b), new type of coronavirus, 2019-nCoV (c), bat SARS like coronavirus CoV SL-CoVZXC21 and bat coronavirus HKU3-1 (d) are shown. The detailed amino acid interaction sites between these two proteins are shown in (e). Arrows showed the areas with interacted residues from both proteins. Amino acid highlighted with different colors indicated the potential interaction residues between different proteins, which was highlighted with different colors.\n\n\nDiscussion\n\nIn this study, we utilized the whole genome sequence of the newly discovered coronavirus, 2019-nCoV, that caused an outbreak of pneumonia in Wuhan, China to perform comparative genetic and functional analysis with the human SARS virus and coronaviruses recovered from different animals. Phylogenetic analysis of coronavirus of different species indicated that 2019-nCoV might have originated from bat, but the intermediate transmission vehicle is not known at this stage. Genetic linkage analysis showed that 2019-nCoV lied at the interface between bat SARS like coronavirus and bat coronavirus and should belong to a novel type of bat coronavirus owing to high degree of variation from the human SARS virus. Analysis of the potential interaction of RBD of 2019-nCoV with human ACE2 receptor protein indicated that its affinity to human cell is much lower than that of human SARS virus due to the loss of several important interaction sites, implying that the infectivity and pathogenicity of this new virus should be much lower than the human SARS virus. These data were the most comprehensive scientific data that support the origin of this new virus, which is important for the following research to identify the intermediate transmission vehicles most likely wild animals. Most importantly, our data supported that the pathogenicity of 2019-nCoV is much lower when compared to SARS virus. These data are very important for the current prevention and treatment of infections caused by this new virus. First, the lower pathogenicity of this new virus may suggest that the infectious dose should be large and the incubation time should be longer, which imply that 1) human to human transmission should mainly happen with close contact within a certain period of time; 2) the longer incubation time should favor the transmission of this virus in human, which suggest that patients with no or mild symptom should also be isolated to prevent further transmission; 3) supportive treatment should be enough to save lives of severe cases. Second, the lower pathogenicity of the virus may suggest that most of the patients should have mild symptom, but they are infectious. Appropriate measures should be designed to isolate these patients without occupying large amount of hospital resources. Thirdly, the lower pathogenicity of the new virus may suggest that it adapts to human much better, which imply that the virus may not disappear after few generations of transmission and long term battle with this virus should be prepared. Lastly, the epidemiology of the outbreak in the early stage is very different from the current dramatic situation. This may not exclude that the virus may have undergo human adaption and mutation in the past month. Using the analysis platform that we have developed above, we should be able to predict whether the new mutations could lead to the increase of infectivity of the mutated virus in a very short time.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended data for 2019-nCoV. https://doi.org/10.6084/m9.figshare.1184822422\n\nThis project contains the following extended data:\n\nExtended+data_F1000Research.docx (Document containing supplementary table and figures)\n\nall-virus-accessions.xlsx (Excel spreadsheet of virus accession numbers used for analysis)\n\nspike-protein-accessions.xlsx (Excel spreadsheet of spike protein accession numbers used for analysis)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nWe are grateful for the discussion and comments from Prof. Mengsu Yang from CityU of Hong Kong. We acknowledge the use of the genome sequence of 2019-nCoV (deposited in GenBank with the accession number MN908947) and other coronavirus sequences that we have used in this study in GenBank. An earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.01.20.913368).\n\n\nReferences\n\nNishimura Y, Yoshida T, Kuronishi M, et al.: ViPTree: the viral proteomic tree server. Bioinformatics. 2017; 33(15): 2379–2380. PubMed Abstract | Publisher Full Text\n\nBah T: Inkscape: guide to a vector drawing program[M]. Upper Saddle River, NJ USA: Prentice Hall. 2011. Reference Source\n\nKumar S, Stecher G, Li M, et al.: MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018; 35(6): 1547–1549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarkin MA, Blackshields G, Brown NP, et al.: Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23(21): 2947–2948. PubMed Abstract | Publisher Full Text\n\nLi F, Li W, Farzan M, et al.: Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005; 309(5742): 1864–1868. PubMed Abstract | Publisher Full Text\n\nMadeira F, Park YM, Lee J, et al.: The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019; 47(W1): W636–W641. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYuan Y, Cao D, Zhang Y, et al.: Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nat Commun. 2017; 8: 15092. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWaterhouse A, Bertoni M, Bienert S, et al.: SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46(W1): W296–W303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu R, Zhao X, Li J, et al.: Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020; pii: S0140-6736(20)30251-8. PubMed Abstract | Publisher Full Text\n\nJi JS: Origins of MERS-CoV, and lessons for 2019-nCoV. Lancet Planet Health. 2020; pii: S2542-5196(20)30032-2. PubMed Abstract | Publisher Full Text\n\nLee PI, Hsueh PR: Emerging threats from zoonotic coronaviruses-from SARS and MERS to 2019-nCoV. J Microbiol Immunol Infect. 2020; pii: S1684-1182(20)30011-6. PubMed Abstract | Publisher Full Text\n\nLi Q, Guan X, Wu P, et al.: Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N Engl J Med. 2020. PubMed Abstract | Publisher Full Text\n\nRothe C, Schunk M, Sothmann P, et al.: Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany. N Engl J Med. 2020; 382(10): 970–971. PubMed Abstract | Publisher Full Text |\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\n\nLau SK, Woo PC, Li KS, et al.: Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A. 2005; 102(39): 14040–14045. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTo KK, Hung IF, Chan JF, et al.: From SARS coronavirus to novel animal and human coronaviruses. J Thorac Dis. 2013; 5 Suppl 2: S103–108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGe XY, Li JL, Yang XL, et al.: Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013; 503(7477): 535–538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi W, Moore MJ, Vasilieva N, et al.: Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003; 426(6965): 450–454. PubMed Abstract | Publisher Full Text\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. PubMed Abstract | Publisher Full Text\n\nDominguez SR, Shrivastava S, Berglund A, et al.: Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses. J Gen Virol. 2014; 95(Pt 4): 836–848. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu D, Zhu C, Ai L, et al.: Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats. Emerg Microbes Infect. 2018; 7(1): 154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen S: Extended data for 2019-nCoV. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11848224.v1"
}
|
[
{
"id": "61587",
"date": "07 Apr 2020",
"name": "Fikret Aydemir",
"expertise": [
"Reviewer Expertise Virology",
"Biochemistry",
"Molecular Biology",
"Vaccine 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\nDong et al. examined the available sequences of coronaviruses to depict the origin and the pathogenicity of 2019-nCoV that caused the current global pandemic. While the data presented in the manuscript could be of interest, several concerns need to be addressed. These concerns presented below.\nMajor:\nMain findings in this study suggest that the new virus should theoretically not be able to cause very serious human disease as compared to other coronaviruses. The mechanism(s) of lowering the pathogenicity, increase human adaptation, or increase incubation times as compared to other strains are not explained, addressed, or cited.\n\nThe epidemiological data are not explained, addressed, or cited.\n\nThe reference for accession # MN908947, the very main isolate obtained from Wuhan, is repeatedly not cited.\n\nThe center that obtained the isolate is not mentioned or cited.\n\nWhat is the exact sequence of the whole genome? Where is it referenced?\n\nClear English writing/editing is needed throughout the paper.\n\nThe limited human transmission potential needs to be explained and cited.\n\nThe intermediate transmission vehicle for other coronaviruses, if exists, needs to be explained and cited.\n\nThe relationship between sequence information and lower pathogenicity needs to be explained and cited.\n\nThe relationship between sequence information, receptor binding, human adaptation, and mutations needs to be explained and cited.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "65534",
"date": "07 Jul 2020",
"name": "Noam Auslander",
"expertise": [
"Reviewer Expertise Computational Biology",
"Bioinformatics",
"Computer science",
"Systems Biology"
],
"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 study by Dong et al. the authors perform a genomic, phylogenetic and structural analysis of SARS-CoV-2, the causal agent of COVID-19. While the goal of the study is worthy and timely, the conclusions drawn are not justified or supported, possibly due to incomplete data or suboptimal methodology that was used throughout this study. The following concerns should be addressed:\nData: the 211 complete genomes of Coronaviridae family that has been collected seem to be only a subset of the available data. There are thousands of complete genomes of viruses from the Coronaviridae family, and substantially more than 211 complete genomes of human CoVs. The selection of these specific 211 should be explained and justified.\n\nAlignment and phylogenetic tree software: The software used to construct alignments and phylogenetic trees is not standard. The authors draw critical conclusions from the results of these applications. However, it is not clear how accurate are the alignments and trees built using the ViPTree tool for closely related viruses within one particular viral family. To draw conclusions from this analysis, the authors should use standard multiple sequence alignment software, such as MUSCLE or MAFFT, and standard phylogenetic tree construction, such as phyML.\n\nIt is unclear how the “Ten spike protein sequences which were similar to that of 2019-nCoV” were selected. What is the criterion used for similarity? Why these particular ten?\n\nThe conclusions drawn appear to be in contradiction to recent literature. The authors write “Based on currently available information, the 2019-nCoV virus belongs to a category that can cause severe illness in some patients but does not transmit readily between people”. However, the references that they cite do not support this notion.\n\nThe authors write “Phylogenetic analysis of genomic sequences of coronaviruses deposited in the GenBank revealed that 2019-nCoV belonged to betacoronavirus and exhibited the closest linkage with two SARS-like coronavirus from bat (bat-SL-CoVZX45 and bat-SL-CoVZX21)”. First, these genomes could not be found in NCBI. I assume that there was a typo, and the authors meant “bat-SL-CoVZXC21” and “bat-SL-CoVZC45”. However, these are not the current closest relatives of SARS-CoV-2, but MN996532 (RaTG13). This may be due to missing sequences or issues with the alignments/tree constructions that are mentioned before. As this is critical to the analysis, it should be revised and addressed.\n\nThe authors conclude that the new coronavirus originated from bat because all coronaviruses that exhibit close linkage with 2019-nCoV originated from bat. However, this may be due to the incomplete data that is used, or other issues, and does not suffice to conclude that SARS-CoV-2 originated from bats. Moreover, many recent studies explored the origin of SARS-CoV-2 in depth. The authors should cite these and compare against their results and conclusions.\n\nThe authors write “Since the S protein is the protein that exhibits the highest degree of genetic variations among different coronaviruses” Is that true? It needs to be cited or supported with data.\n\nThe authors conclude that the infectivity and pathogenicity of SARS-CoV-2 should be much lower than that of SARS-CoV, and that its incubation time should be longer. However, this too seems to contradict most recent studies, which show that the infectivity is not lower (and may be higher) than that of SARS-CoV, and most studies suggest a similar incubation period for SARS-CoV and SARS-CoV-2. These discrepancies should be addressed, and the conclusions supported by recent literature.\n\nThe authors further write that “Analysis of the potential interaction of RBD of 2019-nCoV with human ACE2 receptor protein indicated that its affinity to human cell is much lower than that of human SARS virus“, again, there is a lot of recent work exploring the interaction of RBD of SARS-CoV-2 with human receptors, which seem to report contradicting evidence. This authors should discuss the differences and compare to recent literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 2
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https://f1000research.com/articles/9-121
|
https://f1000research.com/articles/8-2130/v1
|
20 Dec 19
|
{
"type": "Research Article",
"title": "Norovirus genogroup correlation with acute diarrhea severity in Indonesian pediatric patients aged 1-60 months: a cross-sectional study",
"authors": [
"Alpha Fardah Athiyyah",
"Katsumi Shigemura",
"Koichi Kitagawa",
"Nazara Agustina",
"Andy Darma",
"Reza Ranuh",
"Dadik Raharjo",
"Toshiro Shirakawa",
"Masato Fujisawa",
"Subijanto Marto Sudarmo",
"Alpha Fardah Athiyyah",
"Koichi Kitagawa",
"Nazara Agustina",
"Andy Darma",
"Reza Ranuh",
"Dadik Raharjo",
"Toshiro Shirakawa",
"Masato Fujisawa",
"Subijanto Marto Sudarmo"
],
"abstract": "Background: The objective of this study was to investigate the correlation between norovirus genogroup and severity of acute diarrhea in pediatric patients at the Dr. Soetomo Hospital, Surabaya, Indonesia. Methods: This cross-sectional study involved 31 participants aged 1-60 months admitted to the hospital with acute diarrhea from April 2012 to March 2013. Norovirus genogroups (GI and II) were identified from patient stool using reverse transcription polymerase chain reaction (RT-PCR). Severity was measured using the Ruuska and Vesikari scoring system. Results: In total, 91 stool samples were obtained, of which 31 (19%) were norovirus positive. Norovirus GI was found in one sample with mild diarrhea. Norovirus GII was found in 30 samples (96.8%); one sample with mild diarrhea (3.3%), 20 samples with moderate diarrhea (66.7%), and nine samples with severe diarrhea (30%). Conclusion: Norovirus GII was the most prevalent cause of acute diarrhea and 30% of the cases manifested as severe diarrhea.",
"keywords": [
"Diarrhea",
"Infection",
"Norovirus",
"Vesikari score"
],
"content": "Introduction\n\nDiarrhea is considered the second-leading cause of death in pediatric patients under the age of five with a worldwide annual mortality of 525,000 children. Diarrhea lasting for even a few days causes dehydration1. Viruses from the genus Norovirus from the family Caliciviridae are the second-leading cause of acute diarrhea after rotavirus in all age groups of pediatric patients2–4. Norovirus is responsible for 218,000 pediatric deaths (<15 years old) every year and for 1.1 million pediatric hospitalizations around the world2 and was found in 30% of 102 children aged 0–15 months in Jakarta, Indonesia5 and in 19% of 340 children aged 0–60 months in the Dr. Soetomo General Hospital Surabaya, Indonesia6.\n\nEarly identification of norovirus strain and genotype is vital for predicting the development of the disease and selecting the most suitable treatment. Genogroup diversity can be checked using several methods including immunochromatography, reverse transcriptase polymerase chain reaction (RT-PCR), and electron microscopy. Norovirus is grouped into 40 viral strains, which are further classified into five different genogroups. Among these, GI and GII possess the most diverse genetic components7. From the previous study, Norovirus GII.2 genotypes had been the most prevalent norovirus strain in Indonesia (71.4%) followed by norovirus GII.17 (14.3%), one case was GII.4 and one case was GII.18.\n\nNorovirus commonly causes mild and short-term diarrheal episodes9. Nonetheless, this virus can be fatal, particularly in pediatric, geriatric, and immunocompromised patients9,10. Norovirus patients showed severer diarrhea compared to those without norovirus infection in pediatric patients. The type of norovirus strain and genome is thought to be related to diarrhea severity10.\n\nThis paper aimed to examine the correlation between norovirus genogroup and acute diarrhea severity in pediatric patients aged 1–60 months in the Dr. Soetomo General Hospital, Surabaya, Indonesia.\n\n\nMethods\n\nThe study protocol was approved by the Ethical Research Commission of Dr. Soetomo General Hospital, Surabaya, and conducted in line with the 1964 Helsinki declaration and its later amendments or research code of ethic issued by the Ministry of Research, Technology and Higher education. Written informed consent regarding participation in this study, the right to resign, stool and data collection and confidentiality of patient data was obtained and signed from all individuals’ parents. Consent was requested from the patients’ parents because the patients were 1–60 months old.\n\nThis cross-sectional study was conducted between April 2012 and March 2013 of all children aged 1–60 months old with acute diarrhea (described as defecation more than three times per day with change of stool consistency to loose or watery) admitted to the pediatric ward. Patients with a gastrointestinal-anatomical disorder such as Hirschsprung disease, severe systemic disease including sepsis, central nervous system infection or bronchopneumonia, a malabsorption disorder such as cow’s milk allergy, or a compromised immune status were excluded from the study to avoid any bias. On the day of the patients’ admission to the pediatric ward, parents were asked to participate in this study, and they agreed by signing the informed consent form. Stool samples were collected within 24 hours of patient admission with a sterile pot; approximately 3g of stool sample was taken from the middle part of the stool and delivered in no longer than three hours to the laboratory institution. Using a total sampling method, all samples collected until the end of March 2013 were studied.\n\nAll subjects underwent physical examination and the participant’s parents completed a questionnaire.\n\nThe patient assessment was carried out by the physician. The patient’s parents completed questions in the questionnaire regarding characteristic patient data11. These data were: patient’s identity (age, gender and nutritional status); parent’s identity (maternal education); history of diarrhea, which were divided into diarrhea duration (≤4 days, 5 days, and ≥6 days) and diarrhea frequency within 24 hours (1–3 times/day, 4–5 times/day, and ≥6 times/day); vomiting history, divided into vomiting duration (no vomiting, 1 day, 2 days, and more than 3 days) and vomiting frequency within 24 hours (no vomiting, 1 time/day, 2–4 times/day, and more than 5 times/day); and history of breastfeeding (not received breastfeeding, breastfeeding <6 months, and breastfeeding ≥6 months).\n\nAll patients also underwent physical examination of axillary body temperature (°C), arterial pulse measured with a pulseoxymeter (times/minute), respiratory rate (times/minute) and inspection of the signs of dehydration based on WHO classification12 and all results were written down in the questionnaire form. The questionnaire was then reviewed by the researchers and entered into the research database.\n\nStool samples were delivered to the laboratory institution and kept in a deep freezer at -80°C until they were thawed at room temperature prior to RT-PCR analysis. A 10% stool suspension was prepared for each sample prior to RNA extraction by mixing 100µl stool sample with 100µl phosphate buffered saline buffer (Sigma-Aldrich, Germany) with a vortex mixer (QL System, MX-2500 Vortex Mixer, UK) for 15 seconds and then centrifuging at 13,000-15,000 rpm for 10 minutes (Microfuge 20, Beckman Coulter, Indiana Polis, US). The supernatant (1µl) from the stool suspension was transferred into a clean test-tube and the Viral Nucleic Acid Extraction Kit II (Cat # VR100, Geneaid Biotech Ltd., New Taipei, Taiwan) was used to extract viral RNA, carried out according to the kit manufacturer’s instructions. The eluted RNA from the samples was then stored in a deep freezer at -80°C until RT-PCR processing.\n\nReverse transcription was performed by mixing 75 picomoles of pdN6 random hexamers (Cat # 11034731001, Roche Molecular Biochemicals, Germany), 4U AMV Reverse Transcriptase (Cat # AMS.AMV007-1, AMS Biotechnology, Abingdon, UK) and 5μl of the eluted RNA and incubating at 42°C for 60 minutes. Approximately 10μl of the previous mixture was added to 5μl distilled water (RPI, USA), 3μl Ex Taq DNA Polymerase (RR001B, Takara Bio Inc, Japan) and 2μl of both forward and reverse primer. The primer pair used in this study for G1 were the G1SKF primer with nucleotide chain CTGCCCGAATTYGTAAATGA targeting nucleotide position 5342–5362 and a product size of 329 bp, and the G1SKR primer, with nucleotide chain CCAACCCARCCATTRTACA targeting nucleotide position 5652-5671 and a product size of 329 bp. The primer pair used in this study for G2 were the G2SKF primer, with nucleotide chain CNTGGGAGGGCGATCGCAA targeting nucleotide position 5058-5077 and a product size of 344 bp, and G2SKR, with nucleotide chain CCRCCNGCATRHCCRTTRTACAT targeting nucleotide position 5378-5401 and a product size of 344 bp.\n\nPCR reaction was performed as follows. Initial denaturation was done at 94°C for 7 minutes, followed by 40 amplification cycles with Takara PCR Thermal Cycler Dice (TP600, Takara Bio Inc., Japan). Each cycle consisted of denaturation at 94°C for 30 seconds, primer annealing at 50°C for 30 seconds for G1 or 57°C for 30 seconds for G2, extension reaction at 72°C for 45 seconds, followed by a final extension for 2 hours 24 minutes. The PCR product was then separated via gel electrophoresis in a 2% agarose gel and visualized under the UV light after ethidium bromide staining. The gel patterns were captured with Printgraph Fx Series (AE-6933FXN, Atto Corporation, Tokyo, Japan). The RT-PCR method used in this study was the method used by Rasanen et al. in Finland for identifying norovirus13, which can reveal the genotype variety via nonstructural proteins within the virus14. RT-PCR is considered to have the highest sensitivity for diagnosing norovirus infection compared to other methods.\n\nThe severity of diarrhea was measured using the Vesikari Scoring System (see Table 1). This severity scale was originally developed to evaluate the effectiveness of rotavirus vaccines based on 20 points15. The used parameters have been tested for reliability and validity in a cohort study conducted by Freedman with Cronbach’s α > 0.7.\n\nMaximum score = 19; score <7 = mild severity; score 7–10 = moderate severity; score >10 = severe severity.\n\nThis table has been reproduced with reference to the study of Ruuska & Vesikari, 199019\n\nDiarrhea severity was assessed by evaluating seven clinical symptoms, including the duration of diarrhea, diarrhea frequency within 24 hours, vomiting duration, vomiting frequency within 24 hours, body temperature, dehydration status, and treatment. From those components, we could use the modified Vesikari score16 to assess diarrhea severity. Mild diarrhea is equal to a score of < 7; a score of 7–10 is equivalent to moderate manifestation, and severe manifestation scores > 10.\n\nDescriptive analysis was used to determine proportions from patients’ and parents’ identity data (age, gender, nutritional status and maternal education variables) and clinical patient data (diarrhea type, diarrhea duration, diarrhea frequency, vomiting duration and frequency, temperature, dehydration status and causative pathogen). The results of basic and clinical data are presented in tables and divided based on the PCR norovirus result (positive norovirus group and negative norovirus group). The Chi-square test was used to compare diarrhea severity between norovirus genogroups. All collected data were analyzed using SPSS versions 20.0 for windows.\n\n\nResults\n\nSamples were collected in the pediatric wards of the Dr. Soetomo General Hospital Surabaya. A total of 94 stool samples were acquired from eligible subjects within 11 months between April 2012 and March 2013. Of those samples, 31 were positive for norovirus infection using the RT-PCR method (Figure 1)11.\n\nThe basic characteristics of all patients participated in this study are presented in Table 2. Most of the participants whose samples were positive for norovirus were male (54.8%), the youngest participant was one month old and the oldest was 24 months old. Twenty-two participants (71%) were between 6–23 months old. As for nutrition status, most of the subjects had adequate nutrition status (67.7%), while 10 subjects (32.3%) were considered malnourished. A total of 26 subjects (83.9%) were breast fed, with 19 subjects (61.3%) breast fed for more than six months and the rest (22.6%) were breast fed for under six months.\n\nMost patients in negative norovirus group were within 6–23 months old (74.6%) and were male (65.1%). Differences were found in the nutritional status of norovirus negative patients, in whom malnutrition was more prevalent (77.8%) than in norovirus positive patients. Breastfeeding for less than six months was also more common in the norovirus negative group (74.6%).\n\nClinical characteristic data are presented in Table 3. On average, the subjects were brought to the hospital after suffering diarrhea for two days with a frequency of diarrhea of five times within 24 hours. Other symptoms experienced by the subjects included vomiting (71% in positive norovirus group and 63% in negative norovirus group) with the most frequent duration of vomiting being one day (14.9% in positive norovirus group and 40.4% in negative norovirus group) and the most commonly observed frequency of vomiting being 2–4 times (9.6% in positive norovirus group and 21.3% in negative norovirus group) per day. The most frequent body temperature on admission to the hospital was below 37°C for positive norovirus group (48.4%) and sub-febrile (37.1-38.4°C) for the negative norovirus group (50.8%). Dehydration status in this study showed that two of the patients suffered from severe dehydration in both groups, while no dehydration was found only in negative norovirus group (3.2%). Watery stool diarrhea was the most frequent type of diarrhea in the positive norovirus group and negative norovirus group (80.6% and 50.4%, respectively). Bloody or mucoid stool was found only in patients of the negative norovirus group (both 9.5%).\n\nBased on norovirus genogroup identification from gel electrophoresis (Figure 2), GI was found in one sample and GII in 30 samples (96.8%).\n\nA. Negative control lines (NC), marker lines (M), DNA stepladder marks (100bp). Second lines (GI, 329bp); arrow shows 300 bp marker. B. Negative control lines (NC), Marker lines (M), DNA stepladder marks (100bp). Sixth, seventh, and eighth lines (GII, 343bp); arrow shows 300 bp marker.\n\nDistribution between norovirus genogroups and degree of diarrhea severity is presented in Table 4 and shows that norovirus GI was only responsible for one case of diarrhea with moderate severity. From 30 samples that tested positive for norovirus GII, GII was responsible for 20 cases (66.7%) of diarrhea with moderate severity, and nine cases (30%) of diarrhea with severe manifestation.\n\n\nDiscussion\n\nNorovirus has been reported to be the main cause of acute diarrhea worldwide after rotavirus in all age groups of pediatric patients both in developed and developing countries2,3. Norovirus strain type and genome mutation are thought to correlate with the severity of the diarrhea9. It is important to clarify the pathogenesis of this disease to achieve better treatment for each case.\n\nNorovirus was identified in this study in 31 out of 94 samples, with norovirus GII in 30 samples (96.8%) and norovirus GI in one sample (3.2%). This agrees with a previous study done in healthy subjects in Surabaya, Indonesia, which found that all norovirus strains causing gastroenteritis in Surabaya turned out to be GII genotype8.\n\nAnother study conducted in Rio de Janeiro in Brazil from 2005–2008 showed similar results; 1,087 stool samples obtained from 879 people below 20 years old and 208 people above 20 years old had norovirus in approximately 35% of the samples, and 96% of the norovirus-positive samples were GII positive18. A study of 165 participants in Shanghai, China, also showed high prevalence of norovirus GII infection (97.6%), with only 2.42% of those samples positive for norovirus GI8,18,20. These worldwide reports suggest that the most prevalent genogroup infecting humans is GII, with GI only seen in a minority of cases.\n\nThe degree of diarrheal severity in subjects infected by norovirus GII was mostly moderate and only 30% were classified as severe. This agrees with a study carried out by Japanese group, Nakagomi et al., confirming that norovirus infection could elicit a similar degree of severity to rotavirus infection21. Similarly, a study in Taiwan showed that norovirus caused mild diarrhea in 30.6%, moderate diarrhea in 43.9% and severe diarrhea in 25.5% of cases using the Vesikari Scoring System. A previous study also found that norovirus GII infection could lead to severe diarrhea and vomiting compared to infections caused by other genogroups.\n\nThe degree of diarrheal severity in subjects infected by norovirus GI could not be compared to the degree of diarrheal severity in norovirus GII since norovirus GI was only detected in one sample, which is not enough for comparison.\n\nAlthough this study achieved its aims, there were unavoidable study limitations. First, our sample size was comparatively small compared to previous norovirus studies in other countries. Secondly, we found no recurrent cases in our study, and therefore we did not analyze the relationship between norovirus genogroup classification and recurrence of diarrhea. Thirdly, since all the patients that participated in this study were all being admitted to the hospital, the treatment criteria are relatively more severe. Nevertheless, our findings largely agree with previous studies in Surabaya, Shanghai, and Rio de Janeiro, as explained in our discussion above8,18,20. Fourth, we did not have data about other pathogens, which might be co-infecting. In addition, we could not classify the norovirus GII into genotype, and then use this genotype to infer the severity of the disease. Finally, this study only categorized norovirus genogroups by RT-PCR. We did not perform gene sequencing or DNA sequencing for norovirus DNA. Future studies will address these limitations.\n\n\nConclusion\n\nThis study demonstrated that norovirus was responsible for 48.4% of diarrhea cases in the study group, and norovirus GII was significantly dominant compared to norovirus GI. As many as 30% of norovirus cases had severe diarrheal manifestation, all of which were caused by norovirus GII.\n\n\nData availability\n\nHarvard Dataverse: Norovirus PCR data set. https://doi.org/10.7910/DVN/KLBUOE11.\n\nThis project contains the following underlying data:\n\n- Norovirus PCR data set detaverse.tab (sociodemographic information and clinical findings for norovirus positive patients)\n\n- Master data – Noro negative (1).tab (sociodemographic information and clinical findings for both norovirus negative and positive patients)\n\n- Original PCR gel images in JPEG format\n\nHarvard Dataverse: Norovirus PCR data set. https://doi.org/10.7910/DVN/KLBUOE11.\n\nThis project contains the following extended data:\n\n- Norovirus Study Questionnaire (ENG).pdf (copy of questionnaire in English)\n\n- Ind-Informed for Consent_norovirus.pdf (copy of questionnaire in Indonesian)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "References\n\nWorld Health Organization (WHO): Skip to Main Content. 2017.\n\nGallimore C, Barreiros M, Brown D, et al.: Noroviruses associated with acute gastroenteritis in a children's day care facility in Rio de Janeiro, Brazil. Braz J Med Biol Res. 2004; 37(3): 321–326. PubMed Abstract | Publisher Full Text\n\nPatel MM, Widdowson MA, Glass RI, et al.: Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg Infect Dis. 2008; 14(8): 1224–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVincent WF: Twenty Years and Still Going Strong! An Overview of Cryptococcal Infections All about This Publication. 2013; 20: 1–13.\n\nSubekti DS, Tjaniadi P, Lesmana M, et al.: Characterization of Norwalk-like virus associated with gastroenteritis in Indonesia. J Med Virol. 2002; 67(2): 253–8. PubMed Abstract | Publisher Full Text\n\nWardhani S: Epidemiologi Klinis Infeksi Norovirus pada Penderita Diare Bayi dan Anak di RSUD Dr. Soetomo. 2014.\n\nZheng DP, Ando T, Fankhauser RL, et al.: Norovirus classification and proposed strain nomenclature. Virology. 2006; 346(2): 312–323. PubMed Abstract | Publisher Full Text\n\nUtsumi T, Lusida MI, Dinana Z, et al.: Occurrence of norovirus infection in an asymptomatic population in Indonesia. Infect Genet Evol. 2017; 55: 1–7. PubMed Abstract | Publisher Full Text\n\nLopman B, Armstrong B, Atchison C, et al.: Host, weather and virological factors drive norovirus epidemiology: time-series analysis of laboratory surveillance data in England and Wales. PLoS One. 2009; 4(8): e6671. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDivision of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention: Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep. 2011; 60(RR–3): 1–18. PubMed Abstract\n\nAthiyyah AF, Shigemura K, Kitagawa K, et al.: \"Norovirus PCR data set\". Harvard Dataverse, V5, UNF:6:81rGsjPqTo4RxHCdFuR/aA== [fileUNF]. 2019. http://www.doi.org/10.7910/DVN/KLBUOE\n\nWHO: Pocket Book of Hospital Care for Children. 2nd ed. Malta: WHO Press. 2013. Reference Source\n\nRäsänen S, Lappalainen S, Salminen M, et al.: Noroviruses in children seen in a hospital for acute gastroenteritis in Finland. Eur J Pediatr. 2011; 170(11): 1413–1418. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDonaldson EF, Lindesmith LC, Lobue AD, et al.: Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations. Immunol Rev. 2008; 225: 190–211. PubMed Abstract | Publisher Full Text\n\nRuuska T, Vesikari T: Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes. Scand J Infect Dis. 1990; 22(3): 259–67. PubMed Abstract | Publisher Full Text\n\nFreedman SB, Eltorky M, Gorelick M: Evaluation of a gastroenteritis severity score for use in outpatient settings. Pediatrics. 2010; 125(6): e1278–e1285. PubMed Abstract | Publisher Full Text\n\nNajafi A, Iranpour D, Najafi S: Epidemiological surveillance of norovirus diarrhea in hospitalized children with acute gastroenteritis in south of Iran. Jundishapur J Microbiol. 2013; 6(4): 7866. Publisher Full Text\n\nFerreira MS, Victoria M, Carvalho-Costa FA, et al.: Surveillance of norovirus infections in the state of Rio De Janeiro, Brazil 2005-2008. J Med Virol. 2010; 82(8): 1442–1448. PubMed Abstract | Publisher Full Text\n\nRuuska T, Vesikari T: Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes. Scand J Infect Dis. 1990; 22(3): 259–267. PubMed Abstract | Publisher Full Text\n\nZeng M, Gong Z, Zhang Y, et al.: Prevalence and genetic diversity of norovirus in outpatient children with acute diarrhea in Shanghai, China. Jpn J Infect Dis. 2011; 64(5): 417–22. PubMed Abstract\n\nNakagomi T, Correia JB, Nakagomi O, et al.: Norovirus infection among children with acute gastroenteritis in Recife, Brazil: disease severity is comparable to rotavirus gastroenteritis. Arch Virol. 2008; 153(5): 957–960. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "57997",
"date": "06 Jan 2020",
"name": "Mohamad S. Hakim",
"expertise": [
"Reviewer Expertise Virology",
"infectious diseases",
"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\nSummary: Athiyyah et al. reported the prevalence of norovirus infection during one year study of acute diarrhea patients <5 years old admitted to Dr. Soetomo Hospital, Surabaya, Indonesia. Reports of norovirus prevalence in children <5 years old with acute diarrhea in Indonesia are still limited. Therefore, this paper is highly important to expand the data of norovirus surveillance in Indonesia.\nComments: However, this paper needs a major revision to improve the way of presenting and discussing the data as I recommend the following:\nThe sample size is too small to perform correlation analysis. Only one GI positively manifested as moderate diarrhea. The authors should then not use “correlation” in the objective (last paragraph of introduction section), as well as in the title, this does not make any sense. The author can change into a more general title, such as “Clinical manifestation of norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study” or “Norovirus infection in children aged less than five years old admitted with acute diarrhea in Surabaya, Indonesia: a cross-sectional study.”\n\nThe authors mention in the introduction: “Genogroup diversity can be checked using several methods including immunochromatography, reverse transcriptase polymerase chain reaction (RT-PCR), and electron microscopy.” Comments: any supporting evidence or reference of this statement? Norovirus genogroup is based on genetic analysis, so somehow it is weird that it can be differentiated based on electron microscopy.\n\nThe authors did not perform comprehensive literature searching of the previous norovirus study in Indonesia. In fact, there are already some “key” publications in this field, but the authors did not include them in this manuscript. Please thoroughly check the referenced papers to improve author statement in both introduction and discussion section1-5.\n\nPlease change “severer” to “more severe” (in introduction section).\n\nIn the methods: Are there any specific reason to exclude <1 month baby in this study?\n\nFigure 2B, lane 1. Is it considered positive or negative? Because it seems a scanty, positive band there. It looks like Figure 2A, lane 2 which is considered as positive for norovirus GI.\n\nDo the authors include positive controls for each PCR reaction? The prevalence of norovirus in this study (about 32%) is higher than that of global prevalence (about 20%) in countries that did not include Rotavirus vaccination in the NIP. So the authors should ensure that a proper and valid PCR assay has been conducted. Please check: Ahmed SM et al. (2014)6.\n\nFigure 1 is not necessary, so please delete it.\n\nThe authors mention: “Of those samples, 31 were positive for norovirus infection using the RT-PCR method”. Please provide the percentage of norovirus-positive samples.\n\nThe authors mention in discussion: “This agrees with a previous study done in healthy subjects in Surabaya, Indonesia…\" This is not a match comparison, because the previous study is in healthy, asymptomatic adult subjects. The authors should check the above papers that I recommended for much better comparison of previous studies in Indonesia.\n\nIn the discussion, the authors should also discuss associated risk factors of contracting norovirus based on Table 2. For example, why did in your population is the most prevalent age of norovirus positive 6-23 months?\n\nThe authors should add discussion about different severity of norovirus infection based on different genotpe of GII norovirus. Although they did not perform genotype identification in this study, the readers of this paper should still be aware that different GII genotypes can cause different severity of clinical manifestations.\n\nIn conclusion, it is mentioned: “This study demonstrated that norovirus was responsible for 48.4% of diarrhea cases in the study group”. Could the authors clarify about the percentage of 48.4%? 31 out of 94 should be 32.9%. It is also not consistent with the abstract (I mention below).\n\nReference no. 6, what journal that publishes this paper? The authors did not mention this. If this is not published in a peer-reviewed journal, it would be better to change the reference with one I recommended.\n\nThe abstract: “This cross-sectional study involved 31 participants” --> please delete “31”. When you design the study, you never know how many patients will participate. “In total, 91 stool samples were obtained, of which 31 (19%) … “ --> please clarify the percentage. Also, is it 91 or 94? Please thoroughly check all the numbers and percentages you mentioned in this paper.\n\nTable 2, change “Malutrition” to “Malnutrition”. Also, please describe in the methods what the criteria to categorize this nutrition status are.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "5185",
"date": "14 Feb 2020",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "RESPONSES We greatly appreciate your complimentary comments and suggestions. 1. We apologize that our sample size is too small, however, from this limited sample still we could explore many things. 2. We apologize that electron microscopy is not a method could be used for genotyping. We will revise the manuscript to: Genogroup diversity can be checked using immunochromatography and reverse transcriptase polymerase chain reaction (RT-PCR). 3. We will check the suggested reference and revise our manuscript accordingly. 4. We will revise the manuscript accordingly. 5. We exclude neonates below 1 month old because of our limitation to involve neonates admitted in the neonatal wing to follow the study. 6. Figure 2B lane 1 is considered positive. 7. It is our limitation that we do not include positive controls for each PCR reaction. 8. We will revise the manuscript accordingly. 9. We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. 10. We will check the suggested reference and revise our manuscript accordingly. Our study result showed higher norovirus infection incidence than previous studies mentioning incidence about 17-21% (Oyofo et al., 2002; Subekti et al., 2002; Nirwati et al., 2019). One of the possible contributing factors is because we do not use positive controls for our PCR. 11. We will revise our manuscript accordingly. Norovirus infection is most prevalent in 6-23 months due to protection from maternal antibodies during breastfeeding for infant < 6 months old. After 2 years of age, cases of norovirus will decline due to acquired immunity (Japhet et al., 2012; Trang et al., 2012; El Qazoui et al., 2014; Mikounou Louya et al., 2019). 12. Previous studies have shown different severity in the clinical manifestation of Norovirus GII. Genogroup of Norovirus GII.4, GII.2, GII.3, GII.6, and GII.7 are associated with higher severity score (Mathew et al., 2019). However, it is also difficult to determine whether genogroups or viral loads that associated with clinical severity (Chan et al., 2015) 13 We involve 94 subjects in this study and 31 subjects (32.9%) were norovirus positive. We will revise our manuscript. 14. We will check the suggested reference and revise our manuscript accordingly. 15. We will revise our manuscript accordingly. 16. We will revise our manuscript accordingly. Nutrition status is based on WHO curve for children aged 1-60 months. We will check the suggested reference and revise our manuscript accordingly. Thank you very much for reviewing our manuscript. ReferenceChan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185.Chan, M. C. W. et al. (2015) ‘Virus Genotype Distribution and Virus Burden in Children and Adults Hospitalized for Norovirus Gastroenteritis, 2012-2014, Hong Kong.’, Scientific reports, 5, p. 11507. doi: 10.1038/srep11507.Japhet, M. O. et al. (2012) ‘Molecular epidemiology of rotavirus and norovirus in Ile-Ife, Nigeria: High prevalence of G12P[8] rotavirus strains and detection of a rare norovirus genotype’, Journal of Medical Virology, 84(9), pp. 1489–1496. doi: 10.1002/jmv.23343.Mathew, S. et al. (2019) ‘Epidemiological, Molecular, and Clinical Features of Norovirus Infections among Pediatric Patients in Qatar’, Viruses, 11(5), p. 400. doi: 10.3390/v11050400.Mikounou Louya, V. et al. (2019) ‘Molecular characterization of norovirus infection responsible for acute diarrhea in Congolese hospitalized children under five years old in Brazzaville, Republic of Congo’, International Journal of Infectious Diseases, 88, pp. 41–48. doi: 10.1016/j.ijid.2019.07.034.Nirwati, H. et al. (2019) ‘Norovirus and rotavirus infections in children less than five years of age hospitalized with acute gastroenteritis in Indonesia’, Archives of Virology, 164(6), pp. 1515–1525. doi: 10.1007/s00705-019-04215-y.Oyofo, B. A. et al. (2002) ‘Enteropathogens associated with acute diarrhea in community and hospital patients in Jakarta, Indonesia’, FEMS Immunology & Medical Microbiology, 34(2), pp. 139–146. doi: 10.1111/j.1574-695X.2002.tb00615.x.El Qazoui, M. et al. (2014) ‘Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco’, BMC Infectious Diseases, 14(1), p. 300. doi: 10.1186/1471-2334-14-300.Subekti, D. et al. (2002) ‘Incidence of Norwalk-like viruses, rotavirus and adenovirus infection in patients with acute gastroenteritis in Jakarta, Indonesia.’, FEMS immunology and medical microbiology, 33(1), pp. 27–33. doi: 10.1111/j.1574-695X.2002.tb00568.x.Trang, N. V. et al. (2012) ‘Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam’, Journal of Medical Virology, 84(2), pp. 290–297. doi: 10.1002/jmv.23185."
}
]
},
{
"id": "57998",
"date": "27 Jan 2020",
"name": "Hirokazu Kimura",
"expertise": [
"Reviewer Expertise Virology and Infectious Diseases."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors studied the correlation between NoV genogroup and severity of acute diarrhea in the pediatric patients with gastroenteritis at the Hospital, Surabaya, Indonesia. NoV was detected in around 20% of the patients. In many cases, GII virus was detected, while GI was detected in one case. Moreover, 30% of the patients showed severe diarrhea. Overall, the manuscript was well written, while I had some minor comments.\nThe authors only examined by RT-PCR method. Did you detect nonspecific PCR products in the amplicons? Please add it in the results.\n\nHow did you prevent laboratory contamination? Please provide it in the revised manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5178",
"date": "14 Feb 2020",
"name": "Katsumi Shigemura",
"role": "Author Response",
"response": "1. The authors only examined by RT-PCR method. Did you detect non-specific PCR products in the amplicons? Please add it in the results.Response: We suggest that non-specific PCR products refer to viruses other than norovirus. If that so, there were no products other than norovirus was found. 2. How did you prevent laboratory contamination? Please provide it in the revised manuscript.Response: To prevent laboratory contamination, our laboratory staff wore complete apparatuses, such as mask, coat, and gloves. RNA extraction was conducted in Bio Safety Cabinet. Before conducting PCR, all containers were disinfected using alcohol."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2130
|
https://f1000research.com/articles/8-1810/v1
|
29 Oct 19
|
{
"type": "Study Protocol",
"title": "Using machine learning techniques to develop risk prediction models to predict graft failure following kidney transplantation: protocol for a retrospective cohort study",
"authors": [
"Sameera Senanayake",
"Adrian Barnett",
"Nicholas Graves",
"Helen Healy",
"Keshwar Baboolal",
"Sanjeewa Kularatna",
"Adrian Barnett",
"Nicholas Graves",
"Helen Healy",
"Keshwar Baboolal",
"Sanjeewa Kularatna"
],
"abstract": "Background: A mechanism to predict graft failure before the actual kidney transplantation occurs is crucial to clinical management of chronic kidney disease patients. Several kidney graft outcome prediction models, developed using machine learning methods, are available in the literature. However, most of those models used small datasets and none of the machine learning-based prediction models available in the medical literature modelled time-to-event (survival) information, but instead used the binary outcome of failure or not. The objective of this study is to develop two separate machine learning-based predictive models to predict graft failure following live and deceased donor kidney transplant, using time-to-event data in a large national dataset from Australia.\n\nMethods: The dataset provided by the Australia and New Zealand Dialysis and Transplant Registry will be used for the analysis. This retrospective dataset contains the cohort of patients who underwent a kidney transplant in Australia from January 1st, 2007, to December 31st, 2017. This included 3,758 live donor transplants and 7,365 deceased donor transplants. Three machine learning methods (survival tree, random survival forest and survival support vector machine) and one traditional regression method, Cox proportional regression, will be used to develop the two predictive models. The best predictive model will be selected based on the model’s performance. Discussion: This protocol describes the development of two separate machine learning-based predictive models to predict graft failure following live and deceased donor kidney transplant, using a large national dataset from Australia.\n\nFurthermore, these two models will be the most comprehensive kidney graft failure predictive models that have used survival data to model using machine learning techniques. Thus, these models are expected to provide valuable insight into the complex interactions between graft failure and donor and recipient characteristics.",
"keywords": [
"Machine learning",
"Risk prediction models",
"Kidney transplant",
"Graft failure"
],
"content": "Introduction\n\nThe prevalence of chronic kidney disease is increasing globally. Along with this increment, the number of patients in end-stage of renal disease (ESRD) and the demand for kidney transplantation, along with other renal replacement therapies, have increased over recent years1,2. Compared with available renal replacement therapies, renal transplantation has dramatically improved the quality of life and the survival rate of patients with ESRD. However, evidence indicate that the health systems around the world, have failed to meet the increasing demand for kidney grafts. This is evident from the growing prevalence of ESRD in the world3. Further, kidney transplants pose a significant cost burden to health systems compared with other treatment modalities, as they consume a large amount of resources.\n\nIt is important that the donor kidneys are transplanted to the most suitable recipients in order to minimise the number of graft failures, and thus minimise the number of patients returning to the already-burdened waiting list4. However, evidence indicates that the incidence of graft failure following kidney transplantation has increased over the years, possibly owing to increased transplantation of kidneys from expanded-criteria donors and donors after cardiac death, who are more prone to graft failure5. Graft failure is associated with prolonged hospital stay and higher health system costs6,7. A mechanism to predict graft failure before the actual transplantation occurs is crucial in this regard. Similar predictive models have been increasingly used in the recent past, and these have assisted clinicians with evidence-based medical decision-making4,8–10. Numerous conventional predictive models are available in the literature to predict the graft loss among kidney transplant patients11–14. Interestingly, novel techniques based on machine learning methods provide the potential to produce more favourable results15.\n\nMachine learning techniques have been used to develop kidney graft outcome-prediction models4,8–10,16. With the exception of the prediction models developed in the United States4,10,17–19, most of the other prediction models have been developed using datasets with less than 1,000 records. However, evidence indicates that large sample sizes lead to better prediction accuracy in machine learning-based prediction models20. The model developed by Akl et al. (2008)8, using 1,900 live donor transplant records from a single urology centre in Egypt, is the only machine learning predictive model available that is based exclusively on live donor transplants, while most of the other models are based either exclusively on deceased donor transplants, or both deceased and living donor transplants. However, evidence indicates that the graft failure rate and the predictors of graft failure significantly differ between live and deceased donor transplants5. Therefore, from a clinical perspective, two separate valid and reliable prediction models would give superior clinical utility.\n\nTime-to-event (survival) information had not been modelled in any of the machine learning based prediction models available in the medical literature. Instead, most have used the binary outcome of failure or not as the outcome variable. Incorporating the timing of the event to the prediction model, could lead to better prediction models.\n\nIn this background. the objective of this study is to develop two separate machine learning-based predictive models to predict graft failure following live and deceased donor kidney transplant, using time-to-event (survival) data in a large national dataset from Australia.\n\n\nProtocol\n\nThis study will evaluate different machine learning methods in predicting kidney graft failure.\n\nThe dataset was provided by the Australia and New Zealand Dialysis and Transplant Registry (ANZDATA). ANZDATA collects and reports the incidence, prevalence and outcome of dialysis treatment and kidney transplantation for patients with end-stage kidney disease across Australia and New Zealand. The retrospective dataset contains the cohort of patients who underwent a kidney transplant in Australia from January 1st, 2007, to December 31st, 2017. This included 3,758 live donor transplants and 7,365 deceased donor transplants.\n\nTwo separate predictive models will be developed for live donor and deceased donor transplants using separate datasets for live and deceased donor transplants.\n\nGraft survival of the most recent kidney transplants will be converted to a binary variable and will be the primary outcome. Patients who died with a functioning graft will not be considered positive for graft failure, but will be included in all models and censored at their death date. The time to the graft failure will be calculated in days from the date of transplantation. If the outcome of interest has not happened within the time period the data is available (2007 to 2017), it will be considered as right-censored with a time from the date of transplantation to the censoring date. In total, n=65 (0.9%) patients in the deceased donor dataset (n=7,365) and n=73 (1.9%) patients in the living donor dataset (n=3,758) have been lost to follow-up. Their records will be right-censored from the last date where the follow-up information is available.\n\nThe data consist of de-identified recipient and donor characteristics of the transplants. In all, 83 possible variables were identified as potential risk factors for graft failure.\n\nThree machine learning methods and one traditional regression method, Cox proportional regression, will be used to develop the two above-mentioned predictive models. The machine learning methods that will be used are: survival tree, random survival forest and survival support vector machine. Thus, each prediction model will be developed using four methods, and the best predictive model will be selected based on the model’s performance, as described in a later section.\n\nModel development is a systematic process which involves five steps, as indicated in Figure 1.\n\nData preparation. Data preparation involves several steps, such as data cleaning, handling text and categorical attributes, and feature scaling.\n\nRetrospective datasets have the inherent property of having missing values, and most machine learning algorithms do not work well with missing values. Depending on the extent the missing values in each of the variables, the decision will be made to either exclude the particular variable, categorise the missing values as a separate category, or use an imputation method to impute the missing values.\n\nMachine learning algorithms work well with numerical arrays compared with text (e.g. the donor’s cause of death: traumatic brain injury, cerebral infarct and intracranial haemorrhage). Thus, text variables will be categorised appropriately; each category will be assigned a numerical variable (e.g., traumatic brain injury will be assigned a ‘1’, cerebral infarct a ‘2’, and intracranial haemorrhage a ‘3’). However, when text variables are converted to numeric categories, the machine learning algorithms assume that two nearby values are more similar than two distant values (e.g. ‘1’ is more similar to ‘2’ than ‘3’). To overcome this, the categorical variables will be dummy coded into nominal categories.\n\nWith a few exceptions, machine learning algorithms do not perform well when numerical input variables are not in the same scale (e.g., donor age ranges from 9 to 87, while the donor serum creatinine value ranges from 3 to 857). The numerical input variables will be standardised to convert them all to the same scale before applying the different machine learning methods. Parameter estimates and plots will be transformed back to the original scale as this will be the most useful scale for clinicians.\n\nCollinearity in the independent variables will be assessed using variance inflation factor (VIF). VIF of more than four will be considered as presence of collinearity and one of the correlated variables will be excluded from the analysis and the models re-run and re-validated.\n\nFeature selection. Feature (variable) selection is the process of selecting the most relevant variables that should be included in the model. Even though having a large number of input variables may potentially produce superior results with high accuracy, in a practical sense it is important that a manageable number of input variables are used in the model. Having too many input variables may compromise the parsimony and practical usefulness of the model in the clinical setting. In particular, since the predictive models might potentially be used in pre-transplant decision making, only variables available before transplantation will be used in developing prediction models.\n\nThree methods will be used for feature selection:\n\n1. Medical literature and expert opinion. Studies done on kidney transplant graft survival will be reviewed to identify the significant predictors of graft survival. The identified list will be validated by clinical experts. The variables identified as potentially important in this step will be included in the predictive models.\n\n2. Principal component analysis. This method of feature selection will be performed using exploratory factor analysis using principal component analysis. Bartlett’s test of sphericity and the Kaiser-Meyer-Olkin measure will be performed to assess the factorability of the data. Factor structure and factor loadings after varimax rotation will be assessed. The selection of factors will be done, depending on the eigenvalues. The factors are considered relevant if the eigenvalues are more than one. The input variables will be observed for their factor coefficients, and more than 0.4 will be considered as well loaded and will be used for the model development. However, variables that have factor coefficients of less than 0.4 for all the factors will be excluded from the model development.\n\nElastic net. Elastic net uses both Lasso and Ridge regression to select features21. These two regularisation methods minimise the sum of squared residuals using L1 and L2 norms to limit the size of coefficients in the model22. The ideal size of the penalty will be selected using cross-validation. A stronger penalty is a tighter “lasso” that means fewer independent variables are selected. We will examine the plot of variable estimates against the penalty term to understand how the independent variables interact.\n\nPossible combinations of the three sets of selected features from the three different feature selection mechanisms (i.e., medical literature and expert opinion, principal component analysis and elastic net) will be considered as input variables for the four methods of predictive models and the four methods of machine learning algorithms. Seven possible combinations are indicated in Table 1. The best set of input variables for each of the predictive models will be selected based on the model’s performance.\n\nML & EO: Medical literature and expert opinion; PCA : Principal component analysis; EN : Elastic net\n\nModel training. During model training the dataset is randomly divided in to two parts: a training dataset and validation dataset. This prevents over-fitting and provides models that are more robust and give more realistic predictions of their prediction accuracy. Several spilt proportions have been used in models in relevant literature, such as 90:10% and 80:20%, with 70:30% being the most common23. Thus, in the present study dataset will be split in to two parts, with 70% of the data to train the model and 30% to validate the developed models. Since the outcome of interest is a survival function, the training dataset will be fitted to following models; Cox proportional regression method, survival tree, random survival forest and survival support vector machine. The R programming package, specifically the packages Survivalsvm, Ranger, Survival and LTRCtrees, will be used to develop all the predictive models24.\n\n1. Cox proportional regression\n\nCox proportional regression method is a semi-parametric model which is often used to explore the relationship between time-to-event data and several explanatory variables. This method assumes that effects of the different variables on survival are constant over time and are additive in a particular scale.\n\n2. Survival Tree\n\nA survival tree is a tree-like structure, where leaves represent outcome variables, i.e. graft failure (1) or no graft failure (0), and branches represent conjunctions of input variables that produced the outcome. Based on the chosen split criterion (survival statistic), a survival tree divides the data (parent node) into two groups (child nodes). The two resulting groups become the new parent nodes and are subsequently divided further into two child nodes based on the characteristics of the input variables.\n\nHyper-parameters will be regularised until the optimal decision tree is created. The hyper-parameters include maximum depth of the decision tree, minimum number of samples a node must have before it can be split, and the minimum number of samples a node must have.\n\nTrees are often useful for identifying important interactions between independent variables. If strong interactions are found by the tree, then these may be added as additional independent variables to the other approaches as this could increase the models’ predictive ability.\n\n3. Random survival forest\n\nRandom forest is an ensemble method in machine learning where multiple unpruned survival trees are constructed via bootstrap aggregation25,26. Each tree predicts a classification independently and the final prediction is made based on the class (i.e. graft failure versus no graft failure) that gets the most “votes”27,28. This method of aggregation of multiple survival trees has several advantages: the prediction is resistant to outliers, less noisy and suitable for small datasets29.\n\nThe following hyper-parameters will be regularised until the optimal prediction is made: number of survival tree classifiers and maximum number of nodes.\n\n4. Survival support vector machine\n\nSurvival support vector machine is a well-suited method to classify complex but small or medium-sized datasets. Survival support vector machine uses hyperplanes to classify different classes and achieves high predictive accuracy when the data is linearly separable (linear kernel function). However, kernel functions (i.e. Gaussian, sigmoid and polynomial) can be used to separate even the non-linearly separable data, linearly30,31.\n\nInitially, the algorithm will be applied using a linear kernel function, and model performance will be assessed using other kernel functions (i.e. Gaussian, sigmoid and polynomial). Depending on the model’s performance, the best kernel function will be selected. Depending on the kernel function selected, the following hyper-parameters will be regularised until the optimal prediction is made: ‘C’ value, Gamma value, degree and coefficient.\n\nEvaluating the model. Performance of each model will be evaluated using model diagnostics, and the best model will be recommended depending on the results. The trained models, as described in the previous step, will be applied to the validation dataset (30% of the data). The prediction performance of each of the models will be assessed using three methods:\n\n1. Concordance index. The concordance index, or C-index, measures the discriminative ability of a survival model. It is defined as the fraction of pairs of patients that the patient who has a longer survival time is also predicted with lower risk score by the model. The range of concordance is between zero and one, with a larger value indicating better performance (and 0.5 indicating discrimination by chance).\n\n2. Discriminative ability using the C-statistics for the censored function. This is the area under the receiver operating characteristics curve (ROC). The ROC curve is plotted with a sensitivity against 1–specificity, where sensitivity is on the y-axis and 1–specificity on the x-axis. The AUC ranges from 0 to 1, and a higher AUC indicates that the model is capable of distinguishing the cases (i.e. graft failure) with non-case (i.e. no graft failure).\n\nThe best performing model will be selected based on the results of the above-mentioned indicators. In an event of a discrepancy between the performance indicators, the results of concordance index will be considered as the main evaluator. We will also use other model checks such as residuals plots and testing for influential values, which may help to guide decision making about the “best” model.\n\nData that will be used to develop the predictive models will be made available under restricted access with the permission from ANZDATA.\n\nActivities of the ANZDATA registry have been granted full ethics approval by the Royal Adelaide Hospital Human Research Ethics Committee (reference number: HREC/17/RAH/408 R20170927, approval date: 28/11/2017). Even though the data is at the individual-level in the registry, only de-identified records are requested for the analysis. All electronic data will be saved with password protection on Queensland University of Technology’s secure server in encrypted folders only accessible to the nominated research staff.\n\n\nDiscussion\n\nThis protocol describes the development of two separate machine learning-based predictive models to predict graft failure following live and deceased donor kidney transplant, using a large national dataset from Australia. The live donor risk prediction model will be the first machine learning based predictive model developed using a large national dataset, and the deceased donor risk prediction model will be the only machine learning based predictive model that used more than 7,000 patient records outside the United States. Furthermore, these two models will be the only two predictive models which used post kidney transplant graft survival data to model using machine learning techniques. Thus, the two predictive models are expected to provide valuable insight into the complex interactions between graft failure and donor and recipient characteristics.\n\nThe dataset necessary for the study was provided by ANZDATA. ANZDATA collects and reports the incidence, prevalence and outcome of dialysis treatment and kidney transplantation for patients with end-stage kidney disease across Australia and New Zealand. This registry started in 1977 and since then all the kidney transplant activities in Australia and New Zealand have been captured in the registry, including the transplants in the private sector. The inclusion of all kidney transplants in Australia and New Zealand, and the availability and longevity of follow-up information have been the strength of this registry32. Thus, this registry has been the source of information for numerous high-impact publications33–35.\n\nThe current study proposes to use four methods, namely: Cox proportional regression, survival support vector machine, random survival forest and survival tree. The best machine learning technique available to develop a predictive model is currently being discussed widely36. Most are of the view that no single technique fits all datasets, and it depends on the complexity of the data37. Thus, it is imperative that different machine learning methods are used to develop predictive models on a single dataset, so that the best could be chosen using validation parameters.\n\nThis project will have some limitations. According to medical literature, there are an abundance of risk factors for graft failure following a kidney transplant. However, the proposed predictive models will be only based on the variables which have already collected by ANZDATA, thus a complete risk factor profile may not be captured. The graft failure is linked to genetic38 and socio-economic factors39 of the transplant population. Thus, generalisability of the proposed models to other settings outside Australia, needs to be assessed further after they have been developed.",
"appendix": "Acknowledgements\n\nWe acknowledge the Australia and New Zealand Dialysis and Transplant Registry and for their support in providing data for this study. We also acknowledge all the renal units and patients across Australia who contribute data to the registry.\n\n\nReferences\n\nWang T, Xi Y, Lubwawa RN, et al.: Chronic Kidney Disease (CKD) in U.S. Adults with Self-Reported Cardiovascular Disease (CVD)—A National Estimate of Prevalence by KDIGO 2012 Classification. Am Diabetes Assoc. 2018; 67(Supplement 1). Publisher Full Text\n\nValley TS, Nallamothu BK, Heung M, et al.: Hospital Variation in Renal Replacement Therapy for Sepsis in the United States. Crit Care Med. 2018; 46(2): e158–e65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarsoum RS: Chronic kidney disease in the developing world. N Engl J Med. 2006; 354(10): 997–9. PubMed Abstract | Publisher Full Text\n\nBrown TS, Elster EA, Stevens K, et al.: Bayesian modeling of pretransplant variables accurately predicts kidney graft survival. Am J Nephrol. 2012; 36(6): 561–9. PubMed Abstract | Publisher Full Text\n\nDecruyenaere A, Decruyenaere P, Peeters P, et al.: Prediction of delayed graft function after kidney transplantation: comparison between logistic regression and machine learning methods. BMC Med Inform Decis mak. 2015; 15: 83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatas AJ, Gillingham KJ, Elick BA, et al.: Risk factors for prolonged hospitalization after kidney transplants. Clin Transplant. 1997; 11(4): 259–64. PubMed Abstract\n\nRosenthal J, Danovitch G, Wilkinson A, et al.: The high cost of delayed graft function in cadaveric renal transplantation. Transplantation. 1991; 51(5): 1115–8. PubMed Abstract\n\nAkl A, Ismail AM, Ghoneim M: Prediction of graft survival of living-donor kidney transplantation: nomograms or artificial neural networks? Transplantation. 2008; 86(10): 1401–6. PubMed Abstract | Publisher Full Text\n\nGreco R, Papalia T, Lofaro D, et al.: Decisional trees in renal transplant follow-up. Transplant Proc. 2010; 42(4): 1134–6. PubMed Abstract | Publisher Full Text\n\nLin RS, Horn SD, Hurdle JF, et al.: Single and multiple time-point prediction models in kidney transplant outcomes. J Biomed Inform. 2008; 41(6): 944–52. PubMed Abstract | Publisher Full Text\n\nMoore J, He X, Shabir S, et al.: Development and evaluation of a composite risk score to predict kidney transplant failure. Am J Kidney Dis. 2011; 57(5): 744–51. PubMed Abstract | Publisher Full Text\n\nFoucher Y, Daguin P, Akl A, et al.: A clinical scoring system highly predictive of long-term kidney graft survival. Kidney Int. 2010; 78(12): 1288–94. PubMed Abstract | Publisher Full Text\n\nTiong HY, Goldfarb DA, Kattan MW, et al.: Nomograms for predicting graft function and survival in living donor kidney transplantation based on the UNOS Registry. J Urol. 2009; 181(3): 1248–55. PubMed Abstract | Publisher Full Text\n\nRao PS, Schaubel DE, Guidinger MK, et al.: A comprehensive risk quantification score for deceased donor kidneys: the kidney donor risk index. Transplantation. 2009; 88(2): 231–6. PubMed Abstract | Publisher Full Text\n\nKaplan B, Schold J: Transplantation: neural networks for predicting graft survival. Nat Rev Nephrol. 2009; 5(4): 190–2. PubMed Abstract | Publisher Full Text\n\nSenanayake S, White N, Graves N, et al.: Machine learning in predicting graft failure following kidney transplantation: A systematic review of published predictive models. Int J Med Inform. 2019; 130: 103957. PubMed Abstract | Publisher Full Text\n\nTopuz K, Zengul FD, Dag A, et al.: Predicting graft survival among kidney transplant recipients: A Bayesian decision support model. Decision Support Systems. 2018; 106: 97–109. Publisher Full Text\n\nKrikov S, Khan A, Baird BC, et al.: Predicting kidney transplant survival using tree-based modeling. ASAIO J. 2007; 53(5): 592–600. PubMed Abstract | Publisher Full Text\n\nGoldfarb-Rumyantzev AS, Scandling JD, Pappas L, et al.: Prediction of 3-yr cadaveric graft survival based on pre-transplant variables in a large national dataset. Clin Transplant. 2003; 17(6): 485–97. PubMed Abstract\n\nvan der Ploeg T, Austin PC, Steyerberg EW: Modern modelling techniques are data hungry: a simulation study for predicting dichotomous endpoints. BMC Med Res Methodol. 2014; 14: 137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZou H, Hastie T: Regularization and variable selection via the elastic net. Journal of the royal statistical society: series B (statistical methodology). 2005; 67(2): 301–20. Publisher Full Text\n\nTibshirani R: Regression shrinkage and selection via the lasso. Journal of the Royal Statistical Society: Series B (Methodological). 1996; 58(1): 267–88. Reference Source\n\nWong NC, Lam C, Patterson L, et al.: Use of machine learning to predict early biochemical recurrence after robot-assisted prostatectomy. BJU int. 2019; 123(1): 51–57. PubMed Abstract | Publisher Full Text\n\nCore Team R: R: A language and environment for statistical computing. R Foundation for statistical computing Vienna. 2013. Reference Source\n\nEfron B, Tibshirani RJ: An introduction to the bootstrap. CRC press; 1994. Reference Source\n\nBreiman L: Bagging predictors. Machine learning. 1996; 24(2): 123–40. Publisher Full Text\n\nPodgorelec V, Kokol P, Stiglic B, et al.: Decision trees: an overview and their use in medicine. J Med Sys. 2002; 26(5): 445–63. PubMed Abstract | Publisher Full Text\n\nMarshall RJ: The use of classification and regression trees in clinical epidemiology. J Clin Epidemol. 2001; 54(6): 603–9. PubMed Abstract | Publisher Full Text\n\nShaikhina T, Lowe D, Daga S: Decision tree and random forest models for outcome prediction in antibody incompatible kidney transplantation. Biomedical Signal Processing and Control. 2019; 52: 456–462. Publisher Full Text\n\nHu X, Wong KK, Young GS, et al.: Support vector machine multiparametric MRI identification of pseudoprogression from tumor recurrence in patients with resected glioblastoma. J Magn Reson Imaging. 2011; 33(2): 296–305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao D, Liu H, Zheng YH, et al.: A reliable method for colorectal cancer prediction based on feature selection and support vector machine. Med Biol Eng Comput. 2018; 1–12. PubMed Abstract | Publisher Full Text\n\nMcDonald SP, Russ GR: Australian registries-ANZDATA and ANZOD. 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A survey on measuring classification complexity. arXiv preprint. arXiv: 180803591.2018. Reference Source\n\nYanagawa B, Algarni KD, Singh SK, et al.: Clinical, biochemical, and genetic predictors of coronary artery bypass graft failure. J Thorac Cardiovasc Surg. 2014; 148(2): 515–20.e2. PubMed Abstract | Publisher Full Text\n\nMolmenti EP, Alex A, Rosen L, et al.: Recipient Criteria Predictive of Graft Failure in Kidney Transplantation. Int J Angiol. 2016; 25(1): 29–38.. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "55930",
"date": "12 Dec 2019",
"name": "Ramkiran Gouripeddi",
"expertise": [
"Reviewer Expertise Biomedical Informatics",
"Translational Research Informatics",
"Machine Learning",
"Data Integration",
"Exposome Informatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCongratulations to the authors for submitting this manuscript. Here are some comments for your consideration:\nThe authors suggest using model time-to-event modeling instead of binary outcomes. Providing references to show that this is beneficial over binary outputs would be useful.\n\nModeling time-to-event would provide continuous output variables, and therefore require regression methods and not classification methods. It is not clear if the time-to-event is being model as binary variable, in which case the novelty is reduced, or the authors plan to use only regression methods. Please clarify.\n\nRegarding “Three machine learning methods (survival tree, random survival forest and survival support vector machine) and one traditional regression method, Cox proportional regression, will be used to develop the two predictive models.”, is this one model live and one model for deceased donors? Changing this here and elsewhere to read as “one model live and one model for deceased donors, respectively” would make this clear.\n\n“However, evidence indicate that the health systems around the world, have failed to meet the increasing demand for kidney grafts. This is evident from the growing prevalence of ESRD in the world3.” These two sentences do not seem related as suggested by the authors.\n\nListing or providing the 83 variables as a supplementary material will be helpful in reproducing methods.\n\nProviding references for many of the methods mentioned (e.g. VIF) would also help.\n\n“Even though having a large number of input variables may potentially produce superior results with high accuracy, in a practical sense it is important that a manageable number of input variables are used in the model.” This might not always be true. The goal is to avoid the curse of dimensionality and have appropriate number of independent variables.\n\nConsider using cross-validation for model evaluation.\n\nHow is survival SVM different from any SVM?\n\nThe authors could consider evaluating the machine learning models with currently used methods in clinical settings as mentioned in the introductory remarks.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No",
"responses": [
{
"c_id": "5275",
"date": "09 Mar 2020",
"name": "Sameera Senanayake",
"role": "Author Response",
"response": "We are grateful for the comments made by the reviewer. They have been extremely useful for us in reviewing our protocol. We have made changes in response to the suggestions of the reviewer wherever possible and have explained our responses in detail below. Comment 1 : The authors suggest using model time-to-event modeling instead of binary outcomes. Providing references to show that this is beneficial over binary outputs would be useful.Response 1 : The benefit of modelling time-to-event information instead of binary information, has been included to the introduction.“Time-to-event (survival) information had not been modelled in any of the machine learning-based prediction models available in the medical literature, to predict graft failure after kidney transplant. Instead, most have used the binary outcome of failure or not as the outcome variable. However, presence of censored observations makes predictions done using this type of prediction models less accurate. Therefore, incorporating the timing of the event to the prediction model could lead to better prediction models(21).”Comment 2 : Modeling time-to-event would provide continuous output variables, and therefore require regression methods and not classification methods. It is not clear if the time-to-event is being model as binary variable, in which case the novelty is reduced, or the authors plan to use only regression methods. Please clarify.Response 2 : The survival models proposed in this paper will model time-to-event data and so consider two outcomes, i.e. time and graft failure (yes/no) (1,2). This uses more information than a simple binary approach, which is statistically better than a simple binary outcome, and it uses more clinically relevant information, as patients and clinicians would like to know how long the graft will last.1Bou-Hamad I, Larocque D, Ben-Ameur H. A review of survival trees. Statistics Surveys. 2011;5:44-71.2Banerjee M, Reyes-Gastelum D, Haymart MR. Treatment-Free Survival in Patients With Differentiated Thyroid Cancer. The Journal of Clinical Endocrinology & Metabolism. 2018 Jul;103(7):2720-7. Comment 3 : Regarding “Three machine learning methods (survival tree, random survival forest and survival support vector machine) and one traditional regression method, Cox proportional regression, will be used to develop the two predictive models.”, is this one model live and one model for deceased donors? Changing this here and elsewhere to read as “one model live and one model for deceased donors, respectively” would make this clear.Response 3 : This point is well noted. Changes have been made to indicate that two separate models, one for live donors and one for deceased donors, will be developed using different data sets Comment 4 : “However, evidence indicates that the health systems around the world, have failed to meet the increasing demand for kidney grafts. This is evident from the growing prevalence of ESRD in the world3.” These two sentences do not seem related as suggested by the authors. Response 4 : Please note that the final stage of chronic kidney disease is called the End Stage Renal Disease (ESRD) and patients in this stage need to either transplant a kidney or undergo dialysis to sustain life. Evidence indicates that the incidence (new patients) of ESRD is increasing around the world and if the supply of donor grafts increases accordingly, the prevalence (all patients) of ESRD is expected to reduce (as they will not be in ESRD anymore). However, the prevalence of ESRD has been increasing over recent years, indicating the demand for kidney grafts has not been met by the health systems.Therefore, the authors believe that these 2 sentences complement each other. Comment 5 : Listing or providing the 83 variables as a supplementary material will be helpful in reproducing methods.Response 5 : Supplementary material indicating all possible variables has been added. Comment 6 : Providing references for many of the methods mentioned (e.g. VIF) would also help.Response 6 : Reference to the methods mentioned in the text has been added. Comment 7 : Even though having a large number of input variables may potentially produce superior results with high accuracy, in a practical sense it is important that a manageable number of input variables are used in the model.” This might not always be true. The goal is to avoid the curse of dimensionality and have appropriate number of independent variables.Response 7 : Thank you for highlighting this. The following changes have been made to the methods section.“Feature (variable) selection is the process of selecting the most relevant variables that should be included in the model. We will carefully select a potentially large set of variables to be used by the feature selection methods in discussion with clinical colleagues. We will use variable selection methods that aim to create a parsimonious set of predictor variables from the larger set using cross-validation. We will reflect on the variables selected with our clinical colleagues to verify that the model makes clinical sense. Since the predictive models might potentially be used in pre-transplant decision making, only variables available before transplantation will be used in developing prediction models.” Comment 8 : Consider using cross-validation for model evaluation.Response 8 : Thank you for highlighting this. The following changes have been made to the methods section.“During model training, the dataset is randomly divided into two parts: a training dataset and validation dataset. This prevents over-fitting and provides models that are more robust and give more realistic predictions of their prediction accuracy. Several spilt proportions have been used in models in relevant literature, such as 90:10% and 80:20%, with 70:30% being the most common(30). Thus, in the present study dataset will be split into two parts, with 70% of the data to train the model and 30% to validate the developed models. Given our large sample size we expect that this approach would produce similar results to multiple cross-validations. However, in live donor transplant sample of around 3,758 we will use cross-validation to estimate the variability in our model evaluation statistics, and if the variability is large (more than 10% of the mean accuracy) then we will use cross-validation for this sample.” Comment 9 : How is survival SVM different from any SVM?Response 9 : Usual SVM can be used only for dichotomous outcomes and does not account to time-to-event information. However, survival SVM accounts for time-to-event data as it considers outcomes, i.e. time and event (1).1 Fouodo CJ, König IR, Weihs C, Ziegler A, Wright MN. Support Vector Machines for Survival Analysis with R. R Journal. 2018 Jul 1;10(1). Comment 10 : The authors could consider evaluating the machine learning models with currently used methods in clinical settings as mentioned in the introductory remarks.Response 10 : Thank you for highlighting this important point. The outputs of different machine learning predictive models will be compared with Kidney Donor Risk Index (KDPI), commonly used to quantify graft failure risk before transplantation."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1810
|
https://f1000research.com/articles/8-1220/v1
|
30 Jul 19
|
{
"type": "Research Article",
"title": "The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle",
"authors": [
"Gholib Gholib",
"Sri Wahyuni",
"Muslim Akmal",
"Muhammad Hasan",
"Muhammad Agil",
"Bambang Purwantara",
"Sri Wahyuni",
"Muslim Akmal",
"Muhammad Hasan",
"Muhammad Agil",
"Bambang Purwantara"
],
"abstract": "Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, we test the reliability of C and T concentration measurements in the serum of Aceh cattle, obtained using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, we evaluate the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C excretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups).\n\nResults: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.05). Concentrations of T in bulls were significantly higher than in cows (p<0.05). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.",
"keywords": [
"aceh cattle",
"enzyme-linked immunosorbent assay",
"cortisol",
"testosterone",
"analytical validation",
"biological validation",
"hormone stability",
"repeated freezing and re-thawing"
],
"content": "Introduction\n\nCortisol (C) and testosterone (T) are steroid hormones. C is produced by the adrenal cortex1, while T is produced by the Leydig cells in the testes2. C has a key role in physiological stress responses. Accordingly, this hormone is commonly used as an indicator of stress3. On the other hand, T plays an important role in male reproduction physiology. Thus, T levels can be used to assess male gonadal function2.\n\nSteroid hormones can be measured by an enzyme-linked immunosorbent assay (ELISA) technique using various samples (e.g., plasma or serum4, urine5, feces6,7, saliva8,9, and hair10). This technique is now widely used, as it is simple, rapid, convenient, relatively inexpensive, requires a lower sample volume, and particularly, as it is free of radioisotope waste11,12. Moreover, the availability of commercial ELISA kits makes it easy to apply this technique. However, because many commercial ELISA kits are usually designed for humans, using them for animals must be done with great caution and measurements are only trustworthy after the validation of the assay13.\n\nIn order to reliably measure C and T concentrations in cattle using a human commercial ELISA kit, the ELISA kit needs to be validated analytically, physiologically or biologically4,14. Analytical validation can be performed by examining the specificity (cross-reactions), sensitivity, precision, and parallelism (linearity) of the ELISA kit1,4. In addition, it is crucial to perform the biological validation of the assay to examine the ability of the assay to differentiate the variation of the hormone concentration based on the physiological conditions of the animals14. The biological validation of T measurements can be achieved by comparing the concentrations of T from individuals of different age (juvenile versus adult), or sex (male versus female) classes4. For C measurements, the comparison of C concentrations of the same animal before and after some known stressful events (eg., capture, translocation, transportation, and agonistic interactions) can be used as a biological validation procedure14.\n\nAnother critical issue for hormone measurements is the repeated freezing and re-thawing of the samples (freeze-thaw cycles) during the storage prior to the analyses15. Freezing the serum or plasma at -20°C or lower is an ideal storage method16. However, power outages frequently occur, particularly in developing countries, and they can last for a few hours up to a day. Manuals of commercial ELISA kits always explicitly warn to avoid freeze-thaw cycles, because repeated freezing and re-thawing of the samples may affect the stability of the hormones in the serum or plasma, for example: insulin in rats17; adrenocorticotropic hormone in humans18; and sex hormone-binding globulin (SHBG), progesterone, estrone, estradiol, and dehydroepiandrosterone sulfate (DHEAS) in humans19,20. Conversely, several studies have reported no effect of repeated freeze-thaw cycles on the stability of hormone concentrations, such as progesterone in female dogs (bitches)21 and DHEAS, C, dihydrotestosterone, T, estradiol, and progesterone in humans22,23. These different results suggest that the stability may depend on the number of cycles, duration of cycles, and temperature during the repeated freezing and rethawing, as well as on the type of hormones measured.\n\nIn 2011, the Ministry of Agriculture of Indonesia declared Aceh cattle to be a native Indonesian genome resource. This type of cattle can adapt well to the tropical environment and is important for meat production. Increasing the Aceh cattle population is very important for the fulfillment of the protein requirements of the human population in the Aceh region. In this respect, the measurement of C and T can be used for informing husbandry management and breeding programs for this animal. In preparation for the first study to monitor reproduction and stress physiology of Aceh cattle, we test the reliability of commercial C and T ELISA kits designed for human serum/plasma to measure C and T concentrations in the serum of Aceh cattle. Moreover, we evaluate the stability of C and T concentrations in the serum of Aceh cattle after exposure to several repeated freeze-thaw cycles.\n\n\nMethods\n\nThe Institutional Committee of Animal Ethics of the Faculty of Veterinary Medicine, Universitas Syiah Kuala approved the use of all experimental animals in this study (Ref: 33/KEPH/VI/2019)24 (see Extended data). All efforts were made to ameliorate harm to the animals, such as: cattle were placed in the clamp cage to ensure quick, easy and safe collection of the blood sample causing minimal distress; blood samples were collected via the jugular vein without sedation, causing minimal distress. During the study, a member of the study team, accompanied by a veterinarian, observed their behavior for signs of excessive distress or sickness. None of the procedures performed in this study resulted in distress, sickness behavior or weight loss.\n\nSamples were collected from 12 adult Aceh cattle (aged two to five years old and weighing 150–300 kg) during two weeks in April 2019. This sample size was calculated based on Federer’s formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of animals. The sample size calculated using this formula is 16, but it was decided that a sample size of 12 animals would be satisfactory for statistical analysis. Six adult males (bulls) were sampled from a smallholder farmer at Darussalam, Aceh Besar, Aceh Province. These bulls were housed in stables, with each bull was separated by a wooden partition (individual housing system). Six adult female (cows) were sampled from a teaching farm (UPT hewan coba), Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh. These cows were housed together in stables (colony housing system). Cattle housing was equipped with feed and water troughs and food and water were consumed by the cattle ab libitum.\n\nBlood samples (around 5 ml per animal) were collected inside on the farm by a veterinarian from the jugular vein using standard operating procedure without sedation. For the biological validation of C, blood samples were collected in the afternoon (16.00 to 18.00), while for the biological validation of T, blood samples were collected in the morning (06.00 to 08.00). Afterward, the blood was allowed to clot at room temperature for between 30 minutes and two hours. Then, the serum was separated from the red blood cells by centrifugation at 1200xg for 10 minutes at 4°C. After that, the supernatant (serum) was immediately transferred to polypropylene tubes (Eppendorf Safe-Lock tubes) and stored at -20°C.\n\nTo test the capability of the commercial C and T ELISA kits in quantifying concentrations of T and C in the serum of Aceh cattle, the commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits produced by DRG Instruments GmbH, Germany, were evaluated using analytical and biological validations. The procedure for assay validation was conducted as described by Rangel-Negrin et al.14 and Gholib et al.4. Briefly, the analytical validation was comprised of parallelism (i.e. running serial dilution of Aceh cattle serum 1:2 to 1:16, assayed together with C and T standards, and comparing the slope of expected dose versus percent bound of diluted Aceh cattle serum with the slope of C and T standards), accuracy ( i.e. adding known quantities of hormone to C and T standards and calculating the percentage of recovery), and precision (i.e., measuring some low-quality controls (QC L) and high-quality controls (QC H) in one microplate to calculate intra-assay coefficients of variation (CV) and some QC L and QC H in several microplates to calculate inter-assay CV. The sensitivity was reported as provided by the manufacturer.\n\nFor the biological validation of C, we examined the effects of transportation on the C excretion. We used five of the Aceh cattle described above (two bulls and three cows). Bulls and cows were transported using different road vehicles (open car) around Banda Aceh at 11.00 a.m for an hour (~40 km/hour). During transportation, cattle were secured using a rope around their neck and bulls and cows were transported using different vehicles. We predicted that C concentrations should be higher following transportation. We collected serum a day before transportation and four hours after transportation. Serum was collected as described above. For the biological validation of T, we compared the concentrations of T from six bulls and six cows and predicted that the T concentration should be higher in bulls.\n\nTo evaluate the stability of C and T concentrations in serum exposed to repeated freeze-thaw cycles, we took seven serum samples (three bulls and four cows) collected from the previous experiment (biological validation of C and T) to be used for the freeze-thaw cycle experiment, in order to avoid several blood sample collections. Each serum sample was then divided into five aliquots and transferred into 1.5 ml micro-tubes (Eppendorf Safe-Lock tubes; total 35 aliquots; 0.2 ml per tube), closed tightly and sealed with parafilm. All aliquots were subsequently stored frozen at -20°C. Later, those aliquots were subjected to the following treatments: (i) aliquots remained frozen at -20°C until the time of hormone analysis as a control group (N=7); and (ii) aliquots were exposed to repeated freeze-thaw cycles for two, four, six, and eight cycles as test groups (N=7 for each group). The serum was thawed for six hours by placing the tube in a room without an air conditioner (mean of temperature 27.6±0.7°C). Afterward, the serum was refrozen for 24 hours prior to re-thawing. After all freeze-thaw cycles were completed, C and T concentrations were measured for all serum samples together.\n\nThe concentration of C was measured using a commercial C ELISA kit (Cat. no. EIA-1887, DRG Instruments GmbH, Germany). The assay utilizes a monoclonal anti-cortisol antibody and C labeled with horseradish peroxidase as an enzyme conjugate. The measurement of C was conducted following the instructions of the manufacturer (DRG diagnostics). In brief, 20 μl of each standard, control, and samples (serum) in duplicate were dispensed with new disposable tips into appropriate wells on a microplate coated with C monoclonal antibody. After that, each microplate well was filled with 200 μl enzyme conjugate, thoroughly mixed, and then incubated for 60 minutes at room temperature. After incubation, the solution in the wells was briskly shaken out and rinsed out with 350 μl diluted washing solution per well four times. Furthermore, 100 μl substrate solution (tetramethylbenzidine) was added to each well of the microplate, which was then re-incubated for 15–20 minutes at room temperature. After that, the enzymatic reaction was stopped by adding 100 μl of stop solution (0.5 M H2SO4) to each well. Finally, absorbance was determined by using an ELISA reader (xMark™ Microplate Absorbance Spectrophotometer, Bio-Rad Laboratories Inc.) at 450 nm. The C concentration was then calculated using the Microplate Manager® 6 Software (Bio-Rad Laboratories Inc.).\n\nThe concentration of T was measured using a commercial T ELISA kit (Cat. no. EIA-1559, DRG Instruments GmbH, Germany). The assay utilizes a mouse monoclonal anti-testosterone antibody and T labeled with horseradish peroxidase as an enzyme conjugate. This assay has been previously validated successfully for measuring T concentrations in Kacang goats4. T measurements were conducted following the instructions of the manufacturer (DRG diagnostics) and as described by Gholib et al.4. In brief, 25 μl of each standard, control, and samples (serum) in duplicate were dispensed with new disposable tips into appropriate wells on a microplate coated with T monoclonal antibody. After that, each microplate well was filled with 200 μl enzyme conjugate, thoroughly mixed, and then incubated for 60 minutes at room temperature. Following this, the microplates were treated and absorbance was measured as described above for C.\n\nNormality distribution of the data was tested using the Shapiro-Wilk test prior to statistical analysis. For the analytical validation, the parallelism between serial dilutions of two selected serum samples and standard curves was determined by a test of equality of two slopes24. For the biological validation, C data before and after transportation showed a normal distribution (p>0.05), whereas T data of bulls and cows did not (p<0.05). A paired t-test was used for C, whereas for T, a Mann-Whitney U test was used. For the freeze-thaw cycles experiments, the proportion of change in C and T concentrations relative to the control was calculated as (an– xn/xn) x 100, where an is the nth sample value in each freeze-thaw cycles series (two, four, six, eight times) and xn is the value at time zero (control) of the nth sample7,15. The C data were normally distributed (p>0.05), whereas the T data was not (p<0.05). Therefore, for C, a one-way repeated measures ANOVA followed by post hoc analysis using the Bonferroni test was conducted, whereas a Friedman repeated-measures ANOVA on ranks was set up to analyze T concentrations7. We used SigmaPlot 11.0 to create graphs and IBM SPSS 20 to carry out the statistical analysis. All statistical tests were two-tailed and the significance level was set at 0.05.\n\n\nResults\n\nThe serial dilution of the selected serum showed displacement curves that were parallel to C and T standard curves (Figure 1, Table 1). The parallelism, dose-response, accuracy, and precision (coefficients of variation/CV of intra-and inter-assay) are presented in Table 1.\n\na) Cortisol (C) enzyme-linked immunosorbent assay (ELISA) kit: sample one (white circle) and sample two (black triangle down) were diluted 1:2 to 1:16 in assay buffer and measured with serial C standards (black circle) ranging from 10 to 400 ng/ml. The curve of sample one, sample two, and C standards produced almost identical R2 values: 0.987, 0.945, and 0.992, respectively. b) Testosterone (T) ELISA kit: sample one (white circle) and sample two (black triangle down) were diluted 1:2 to 1:16 in assay buffer and measured with serial T standards (black circle) ranging from 0.5 to 16 ng/ml. The curve of sample one, sample two, and T standards produced almost identical R2 values: 0.964, 0.993, and 0.945, respectively.\n\na = value in manual protocol of assay from manufacturer\n\nThe mean (± SD) of the C concentrations in the samples post-transportation (37.77 ± 6.01 ng/ml) was significantly higher compared to pre-transportation samples (31.36 ± 4.39 ng/ml; t = -3.403, p = 0.027; Figure 2A). The mean (± SD) of the T concentrations in bulls (4.25 ± 1.78 ng/ml) was significantly higher than the T concentrations in cows (0.68 ± 0.33 ng/ml; U = 0.0005, p = 0.004; Figure 2B). Results on the analytical and biological validations indicated that both C and T ELISA kits were reliable assays for measuring C and T concentrations in Aceh cattle. Raw values of analytical validation results are given in Dataset 125, whereas raw values of biological validation results are given in Dataset 226 (see Underlying data).\n\na) Concentrations of cortisol (C) before (pre-trans) and after (post-trans) transportation measured using a commercial C ELISA kit (DRG, Cat. No. EIA-1887). b) Concentrations of testosterone (T) in bulls and cows measured using a commercial T ELISA kit (DRG, Cat. No. EIA-1559). Different superscripts above line symbols and histogram indicate a significant difference between groups (p < 0.05).\n\nRepeated freeze-thaw cycles in serum significantly affected the stability of C concentrations (F[4,30] = 11.681, p<0.001, N=7; Table 2). Post hoc analysis showed that after four to eight freeze-thaw cycles, the C concentrations were significantly lower than those of the control group (all p < 0.05; Table 2, Figure 3A). In contrast to the C concentrations, the T concentrations remained stable after exposure to two to eight freeze-thaw cycles (χ2[4] = 7.626, p = 0.106, N = 7; Table 2, Figure 3B). The mean percentage of change in C and T concentrations ranged between 13.43 to 33.94% and 3.55 to 8.33%, respectively, relative to the control group (Figure 3). Raw values of C and T concentrations are given in Dataset 327 (see Underlying data).\n\na,b,cDifferent superscripts above value indicate a significant difference between groups (p < 0.05)\n\na) Cortisol (C). b) Testosterone (T). Values represent mean±SD relative to control (100%). Different superscripts above histogram indicate a significant difference between groups (p < 0.05).\n\n\nDiscussion\n\nIn the current study, we demonstrate that C and T concentrations in the serum of Aceh cattle can be accurately measured using the commercial C and T ELISA kits designed for human C and T measurements. The results of the parallelism test show that the slope of the diluted sample curves was parallel to the standard curves of commercial C and T ELISA kits. In addition, concentrations of C and T decreased significantly following the dilution levels. Furthermore, these assays also show high accuracy (~100%) and precision (CV < 10% and < 15% for intra- and inter-assay, respectively). Therefore, both the commercial C ELISA kit and the commercial T ELISA kit tested can be used to measure C and T concentrations, respectively, reliably in the serum of Aceh cattle.\n\nA significant increase in C excretion after transportation was detected by the commercial C ELISA kit tested (Figure 2A). This result was to be predicted because C is the main modulator of physiological stress and it will increase in response to a stressor28. The commercial T ELISA kit also appeared reliable in its ability to discriminate T concentrations between bulls and cows. T concentrations in bulls were more than five times higher compared to cows. This result was to be expected because T is the major androgen produced by the Leydig cells of the testes29, whereas only small amounts are secreted in the adrenal cortex and ovaries of females30.\n\nOur results indicated that the biological validation of commercial C and T ELISA kits can be performed using C measurements in relation to a stressful event and T measurements of different sexes. It is crucial to conduct such as validation to ensure the biological meaningfulness of the analyses31,32. EIA-1887 and EIA-1559 are commercial ELISA kits developed for measuring hormone concentrations, particularly in human serum/plasma. These assays use an antibody highly specific to either C or T. The antigen (C or T) in the serum of Aceh cattle can bind correctly with the antibody (anti-cortisol and anti-testosterone) in these ELISA kits4 because the side-chains of the carbon compounds of C (C-21)33 and T(C-19)30 have similar structures in vertebrates. The validation of commercial ELISA kits for measuring hormones in animals has been successfully conducted in several animals, such as progesterone in cattle34, estradiol and inhibin A in buffalo35, C in horses36, and T in Kacang goats4.\n\nIn addition to the validation of ELISA kits, we tested the effects of repeated freeze-thaw cycles on the stability of C and T concentrations in serum. Concentrations of C decreased significantly after the exposure to four, six, and eight freeze-thaw cycles (p<0.05), while T concentrations did not (p>0.05). Concentrations of C declined up to 33.94% after eight freeze-thaw cycles, whereas the percentage changes in T concentrations were less than 10% in all test groups. These findings show that T is more resilient to freeze-thaw cycles compared to C. The reason why C and T concentrations decreased after exposure to repeated freeze-thaw cycles is not entirely clear. It is possible, however, that the serum left at high temperatures (27.6±0.7°C) during the six hours of the thawing process might facilitate the increased degradation of C and T15. Consequently, less antigen in the serum binds with the antibody of the assay37. Thus, C and T concentrations in all test groups were lower than the control group.\n\nFrom a practical point of view, our results suggest that commercial C (EIA-1887) and T (EIA-1559) ELISA kits can accurately measure C and T concentrations, respectively, in the serum of animals such as Aceh cattle. It can advantageous to use these commercial assays over assays specifically designed for animals because of their relatively low price. On the other hand, repeated freeze-thaw cycles must be considered, especially for the C measurement. Therefore, conditions that may potentially cause repeated freeze-thaw cycles of samples designated for hormone analysis (e.g. frequent power outages) should be avoided. Some efforts can be performed to prevent the possibility of repeated freeze-thaw cycles, such as installing a backup generator in each laboratory to supply electricity when there are power outages, filling each freezer with a blanket of ice packs to maintain the temperature inside the freezer during the power outage, averting the use of an aliquot of serum/plasma for several analyses at different times, and dividing serum into several aliquots when several analyses should be performed from the same sample15.\n\nThis study has some limitations: first, the small sample size used; and second, the information regarding the substantive reason for decreasing C and T concentrations after repeated freeze-thaw cycles is still unclear. To elucidate this reason, the biochemistry of the serum after exposure to freeze thaw-cycles needs to be investigated in future studies. Moreover, it was unclear whether these assays are also reliable for other animals, so they would need a validation test for each animal. Apart from the fact that there are limitations, the results are important to support further studies in Aceh cattle, particularly for maintaining and growing the Aceh cattle population to ensure food security in the Aceh region and Indonesia as a whole.\n\nIn conclusion, our study shows that commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. We also demonstrate that more than two repeated freeze-thaw cycles significantly affected the stability of serum C concentrations, but up to eight repeated freeze-thaw cycles did not significantly affect the stability of serum T concentrations.\n\n\nData availability\n\nFigshare: Dataset 1. https://doi.org/10.6084/m9.figshare.8342504.v225\n\nThis project contains the following underlying data:\n\n- Analytical validation data.csv (raw values of analytical validation results included data of parallelism, accuracy, and precision [coefficients of variation/CV of intra-and inter-assay])\n\nFigshare: Dataset 2. https://doi.org/10.6084/m9.figshare.8342651.v126\n\nThis project contains the following underlying data:\n\n- Data of Biological validation.csv (raw values of biological validation results: cortisol concentrations before and after transportation and testosterone concentrations in bulls and cows of Aceh cattle)\n\nFigshare: Dataset 3. https://doi.org/10.6084/m9.figshare.8342720.v127\n\nThis project contains the following underlying data:\n\n- Data of freeze-thaw cycles on cortisol and testosterone.csv (raw value of cortisol and testosterone concentration in Aceh cattle serum after exposure repeated freeze-thaw cycles)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Extended data 1. https://doi.org/10.6084/m9.figshare.8487830.v124\n\nThis project contains the following extended data:\n\n- Ethical Clearance Approval Gholib et al..pdf (certificate of ethical clearance approval for using animals)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Grant information\n\nThis study was funded by The Ministry of Research, Technology and Higher Education, Indonesia [215/SP2H/LT/DPRM/2019]. This paper also supported by USAID through Sustainable Higher Education Research Alliances (SHERA) Program - Center for Collaborative Research Animal Biotechnology and Coral Reef Fisheries (CCR ANBIOCORE).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe are grateful to Julia A. Kunz for valuable comments, suggestions, and English corrections during preparing this manuscript.\n\n\nReferences\n\nSheriff MJ, Dantzer B, Delehanty B, et al.: Measuring stress in wildlife: techniques for quantifying glucocorticoids. Oecologia. 2011; 166(4): 869–887. PubMed Abstract | Publisher Full Text\n\nHardy MP, Gao HB, Dong Q, et al.: Stress hormone and male reproductive function. Cell Tissue Res. 2005; 322(1): 147–153. PubMed Abstract | Publisher Full Text\n\nMöstl E, Palme R: Hormones as indicators of stress. Dom Anim Endocrinol. 2002; 23(1–2): 67–74. PubMed Abstract | Publisher Full Text\n\nGholib G, Wahyuni S, Kadar OH, et al.: Measurement of serum testosterone in kacang goat by using enzyme-linked immunosorbent assay (elisa) technique: the importance of kit validation. J Ked Hewan. 2016; 10(1): 32–36. Publisher Full Text\n\nDu Z, Keeley T, Janssen T, et al.: Measurement of testosterone and cortisol metabolites and luteinising hormone in captive southern hairy-nosed wombat (Lasiorhinus latifrons) urine. Gen Comp Endocrinol. 2017; 250: 70–79. PubMed Abstract | Publisher Full Text\n\nNugraha TP, Heistermann M, Agil M, et al.: Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans. Primates. 2017; 58(2): 285–294. PubMed Abstract | Publisher Full Text\n\nGholib G, Heistermann M, Agil M, et al.: Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques. Primates. 2018; 59(3): 281–292. PubMed Abstract | Publisher Full Text\n\nDurdiaková J, Fábryová H, Koborová I, et al.: The effects of saliva collection, handling and storage on salivary testosterone measurement. Steroids. 2013; 78(14): 1325–1331. PubMed Abstract | Publisher Full Text\n\nBloomer RJ: Considerations in the Measurement of Testosterone in Saliva and Serum using ELISA Procedures. Br J Med Med Res. 2015; 5(1): 116–122. Publisher Full Text\n\nStradaioli G, Peric T, Montillo M, et al.: Hair cortisol and testosterone concentrations and semen production of Bos taurus bulls. Ital J Anim Sci. 2017; 16(4): 631–639. Publisher Full Text\n\nDorgan JF, Fears TR, McMahon RP, et al.: Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry. Steroids. 2002; 67(3–4): 151–158. PubMed Abstract | Publisher Full Text\n\nSakamoto S, Putalun W, Vimolmangkang S, et al.: Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. J Nat Med. 2018; 72(1): 32–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKinn Rød AM, Harkestad N, Jellestad FK, et al.: Comparison of commercial ELISA assays for quantification of corticosterone in serum. Sci Rep. 2017; 7(1): 6748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRangel-Negrin A, Flores-Escobar E, Chavira R, et al.: Physiological and analytical validations of fecal steroid hormone measures in black howler monkeys. Primates. 2014; 55(4): 456–65. PubMed Abstract | Publisher Full Text\n\nGholib G, Agil M, Supriatna I, et al.: Repeated freeze-thaw cycles but not short-term storage of fecal extracts at ambient temperature influence the stability of steroid metabolite levels in crested macaques. J Ked Hewan. 2017; 11(2): 78–85. Publisher Full Text\n\nHodges JK, Heistermann M: Field Endocrinology: Monitoring Hormonal Changes in Free-Ranging Primates. In: Setchell JM, Curtis DJ (eds) Field and Laboratory Methods in Primatology: A Practical Guide. 2nd edn. Cambridge University Press, Cambridge. 2011; 353–370. Publisher Full Text\n\nBielohuby M, Popp S, Bidlingmaier M: A guide for measurement of circulating metabolic hormones in rodents: Pitfalls during the pre-analytical phase. Mol Metab. 2012; 1(1–2): 47–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHillebrand JJ, Heijboer AC, Endert E: Effects of repeated freeze-thaw cycles on endocrine parameters in plasma and serum. Ann Clin Biochem. 2017; 54(2): 289–292. PubMed Abstract | Publisher Full Text\n\nReyna R, Traynor KD, Hines G, et al.: Repeated freezing and thawing does not generally alter assay results for several commonly studied reproductive hormones. Fertil Steril. 2001; 76(4): 823–825. PubMed Abstract | Publisher Full Text\n\nComstock GW, Burke AE, Norkus EP, et al.: Effects of Repeated Freeze-Thaw Cycles on Concentrations of Cholesterol, Micronutrients, and Hormones in Human Plasma and Serum. Clin Chem. 2001; 47(1): 139–142. PubMed Abstract\n\nTahir MZ, Thoumire S, Raffaelli M, et al.: Effect of blood handling conditions on progesterone assay results obtained by chemiluminescence in the bitch. Domest Anim Endocrinol. 2013; 45(3): 141–144. PubMed Abstract | Publisher Full Text\n\nKley HK, Rick W: The effect of storage and temperature on the analysis of steroids in plasma and blood. J Clin Chem Clin Biochem. 1984; 22(5): 371–378. PubMed Abstract\n\nBauman JE: Stability of radioimmunoassayable steroid and protein hormones after repeated freeze-thaw cycles. Clin Chem. 1982; 28(11): 2336–2337. PubMed Abstract\n\nGholib G, Wahyuni S, Akmal M, et al.: Extended data 1.figshare. Figure. 2019. http://dx.doi.org/10.6084/m9.figshare.8487830.v1\n\nZar JH: Biostatistical Analysis. Prentice Hall, Upper Saddle River, N.J. 1996.\n\nGholib G, Wahyuni S, Akmal M, et al.: Dataset 1. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8342504.v2\n\nGholib G, Wahyuni S, Akmal M, et al.: Dataset 2. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8342651.v1\n\nGholib G, Wahyuni S, Akmal M, et al.: Dataset 3. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8342720.v1\n\nSapolsky RM, Romero LM, Munck AU: How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 2000; 21(1): 55–89. PubMed Abstract | Publisher Full Text\n\nSwerdloff RS, Wang C, Sinha-Hikim AP: Hypothalamicpituitary-gonadal axis in men. In Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT (eds) Hormones, brain, and behavior. 2nd edn. Academic Press, San Diego. 2009; Part V: 2357–2393. Reference Source\n\nChedrese PJ: Reproductive Endocrinology: A Moleculer approach. Springer Science + Madia Business, New York, USA. 2009. Publisher Full Text\n\nHodges K, Brown J, Heistermann M: Endocrine monitoring of reproduction and stress. In: Kleiman DG, Thompson KV, Kirk Baer C (eds) Wild mammals in captivity: principles and techniques for zoo management. The University of Chicago Press, Chicago. 2010; 447–468. Reference Source\n\nHeistermann M, Palme R, Ganswindt A: Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis. Am J Primatol. 2006; 68(3): 257–273. PubMed Abstract | Publisher Full Text\n\nMöstl E, Messmann S, Bagu E, et al.: Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. Zentralbl Veterinarmed A. 1999; 46(10): 621–631. PubMed Abstract | Publisher Full Text\n\nBayemi PH, Nsongka VM, Perera BMAO, et al.: Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon. Trop Anim Health Prod. 2007; 39(5): 335–338. PubMed Abstract | Publisher Full Text\n\nTodini S, Terzano GM, Malfatti A: Validation of ELISA kits for determination of Inhibin-A and Estradiol-17-beta concentrations in Buffalo plasma. Ital J Anim Sci. 2007; 6(sup2): 585–588. Publisher Full Text\n\nPawluski J, Jego P, Henry S, et al.: Low plasma cortisol and fecal cortisol metabolite measures as indicators of compromised welfare in domestic horses (Equus caballus). PLoS One. 2017; 12(9): e0182257. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPappano DJ, Roberts EK, Beehner JC: Testing extraction and storage parameters for a fecal hormone method. Am J Primatol. 2010; 72(11): 934–941. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "52712",
"date": "03 Sep 2019",
"name": "Pudji Astuti",
"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 details comment of the paper are attached (Please see attachment).\n\nOverall the content are complete including the validation test and the Biological validation are conducted.\n\nThe theory of similarity base chemical formula namely cyclopentano perhydro phenanthrene both of human and animal should be added.\n\nThe information standard of parallelism should be added.\n\nAlthough the number of samples is less than specified (based on the Frederer formula), the results can still be accounted for because the amount of 12 samples is still very sufficient considering the large number of replications.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5169",
"date": "04 Feb 2020",
"name": "Gholib Gholib",
"role": "Author Response",
"response": "Response to Reviewer 1.First, we would like to thanks to the reviewer 1 for approved our paper. Her comments and suggestions have helped us to further improve the paper. As you will see in the response-to-reviewer below, we have answered all points raised and have changed the paper accordingly. 1. Do not use personal pronouns. Please don't use we, I etc Response: It has been changed throughout the text.2. Choose the most 5 important words Response: In the guideline, it was mentioned that we can use until 8 keywords. We still keep the keywords because using more keywords will help people to find our paper.3. Do not use abbreviation word on the beginning of the lineResponse: It has been changed throughout the text.4. Is there any correlation between C and T in this case?Response:Yes there is a correlation between cortisol and testosterone in some animals from previous studies. From the previous study, high levels of cortisol can suppres the testosterone production.An additional sentence and references have been added in the revised paper5. Due to the same base form of steroid (cyclopentanoperhydrophenantrend), we can use the same kit for animal as in human.Response: Thank you for your suggestion, This statement has been mentioned in the discussion section.6. Do not use personal pronounsResponse: It has been changed throughout texts 7. Should be done by veterinariansResponse: During the study, our team was accompanied by a veterinarian during data collection.Blood samples were performed by veterinarians. This statement has been written in the paper ( ethical statement, collection and processing of blood samples) 8. How to validate the data, because the samples are less than it should be?Response: We have added the number of samples. Now, 32 Aceh cattle were used: 16 bulls and 16 cows. For the testosterone validation test, all cattle ( 16 bulls and 16 cows) were used. For cortisol validation test, 16 cattle ( 8 bulls and 8 cows ) were used which is taken from 32 Aceh cattle above. The explanation has been added in the revised paper9. Please add some information of the feed and the volume per head.Response: Feed given to the cattle is grass at 10% of body weight and 1% of body weight of rice bran and tofu dregs. The explanation has been added in the revised paper 10. until the assay process are conductedResponse: Thank you for your suggestion, we have added some words in revised paper11. Please add sign of significant on testosteroneResponse: Superskrip “ a” has been added to the testosterone data in the Figure 3, because they showed not significant different. Table 2. has been deleted from the revised paper.12. Due to the same base form, the kit also can be used in animals as in human. Please add this explanationResponse: The explanation has been added in the discussion section, paragraph 3rd in revised paper13. Please explain about gradient slope that is used in the research as standard. Is there prerequisite to determine parallelism standart?Response: To determine whether curve of serial dilution samples was parallel with the curve of hormone standard (C and T), the slope of the serial dilution samples curve must be compare with the slope of the standard curve. For this, we can do a statistical analysis to compare the value of the slope from dilution samples curve and standard curev. We can determine that the both slopes (sample curve and standard curve) were parallel when there was not significant different between slope of dilution sample curve with standard curve. From our results, Slope of Sample one and sample two were not significant different with the slope of the standard curve (p>0.05). It is indicated that both slope are similar.14. Why biology validation should be done? Is parallelism test based on dilution can be confirmed? In the real condition, does the treatment should be validated by biology validation? Does parallelism test not enough?Response: To test the reliability of the aasay, there are several validations should be done namely: analytical validation, physiological validation, and biological validation. Parallellism is one part of the analytical validation. This test aimed to evaluate whether antigen (hormone) can bind with the antibody in the assay accurately and find the optimal dilution if needed. However, parallelism test is not a marker of specificity of the assay, but aproof of a dose–response relationship (Möstl et al., 2005). Therefore, it is not possible to selectthe best-suited assays to assess physiological stress and reproduction only based on the parallelism results. To achieve that, it is important to perform a biological validation or physiological validation. For physiological validation of stress hormone, a widely used method is an adrenocorticotrophic hormone (ACTH) challenge test by injecting animals with ACTH (for review see Tauma & Palme 2005 ) and by injecting radiolabeled testosterone [4(C)-14C testosterone] for testosterone validation (Möhle et al. 2002)."
}
]
},
{
"id": "54435",
"date": "16 Oct 2019",
"name": "Annaïs Carbajal",
"expertise": [
"Reviewer Expertise Endocrinology and animal stress"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 validated an EIA for testosterone and cortisol detection in serum of Aceh cattle. They also performed the biological validation of both hormones in serum. And finally, they evaluated the effect of freeze-thaw cycles on the stability of these hormones in serum.\n\nAlthough the technique they present is not new, the validation the authors perform are imperative for the proper hormone interpretation in every matrix, and thus this should be done for each species, matrix and assay of interest. The study has a robust experimental design despite using a low number of samples in some experiments. Results they present are very useful and may provide guidance for future studies to perform appropriate validations of their methods.\nMy major concern is why the authors discuss that T is affected by the freeze-thaw cycles if results do not indicate the same. Additionally, I would suggest dividing the aims in three: analytical validation, biological validation and freeze-thaw cycles, as they may provide three different results and conclusions. I have also added some minor comments which can be found in the pdf document that I hope they will be useful for the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "5170",
"date": "04 Feb 2020",
"name": "Gholib Gholib",
"role": "Author Response",
"response": "Response to Reviewer 2.First, we would like to thanks to the reviewer 2 for reviewing our paper and gave approved with reservations . Her comments and suggestions have helped us to further improve the paper. As you will see in the response-to-reviewer below, we have answered all points raised and have changed the paper accordingly. We very much hope that the paper is now finally approved.1. New sentence should not start with an abbreviation. This needs to be rectified in the whole textResponse: All abbreviation in new sentences have been changed throughout text.2. T is also produced by the ovaries and the adrenalsResponse: Yes, you are right, T is also produced by the ovaries and the adrenals. An explanation has been added in revised paper3. Could also be due to species-specific differences?Response: Yes, it can be influence by species-specific differences, Thank you very much for the revision. It has been added to the revised paper.4. The aims of the study are not fully clear here. As three different results are discused, I would recommend dividing the aims in 3 clear objectives: Analytical validation, biological validation and freeze-thaw cyclesResponse: Thank you very much for your suggestion. Now, The aims were dividedinto three objectives. The objective of the present study were: First, conducting an analytical validation test to examine the reliability of commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits that accurately measure C and T concentrations, respectively in Aceh cattle; Second, performing a biological validation test to examine the reliability of commercial C ELISA kit (Cat. no. EIA-1887) to the response a stress event by comparing C concentrations before and after transportation and the reliability of T ELISA kit (Cat. no. EIA-1559) ELISA kits in discriminating the T concentrations from different sexes (bulls versus cows). The hypothesis is that C concentrations should be higher following transportation and the T concentration should be higher in bulls. Finally, the stability of C and T concentrations in the serum of Aceh cattle after exposure to several repeated freeze-thaw cycles was evaluated. For details, please see in the revised paper.5. This is not totally true since only 5 individuals are used for the C validation.Response: Thank you for your correction. Thank you for your correction. We have collected additional samples ( 10 bulls and 10 cows) to increase the number of samples. Now, in total 32 Aceh cattle were used: 16 bulls and 16 cows. For the testosterone validation test, all cattle ( 16 bulls and 16 cows) were used. For cortisol validation test, 16 cattle ( 8 bulls and 8 cows ) were used which is taken from 32 Aceh cattle above. The explanation has been added in the revised paper.6. Information is lacking here. For example, why did you collect samples at 16h and 6h? This information should go to the subsection \"biological validation\" - experimental design.Response: For biological validation of C, Samples were collected at 16 h, four hours after the trip to monitor the increase in Cortisol after the transportation. For biological validation of T, Samples were collected in the morning (6h-8h) because in some speceies including cattle, there is diurnal variation of T in blood which concentration of T ishigher in the morning compared to afternoon. This explanation has been added to the subsection of biological validation. 7. Hypothesis are written in the introductionResponse: Hypothesis has been deleted in this section and moved to the subsection “ introduction”.8. Why serum was collected a day before and not immediately before transport? Please, specify the time of day when samples were collected.Response: Sample was collected a day before to prevent the stress in cattle and sometimes blood collection in cattle needs some times. More explanation has been added in the revised paper9.This is mean and standard deviation? Please, specifyResponse: Thank you very much for your correction, it has been added in the revise paper.10. Ref 24 extended data 1 shows: Certificate of ethical clearance approval for using animals for study on monitoring reproduction and stress in aceh cattleData on the test of equality is not provided. Here the reference should be ref 26.Indicate the name of the statistical model used to \"test of equality of two slopes\"Response: Model used for the test of equality of two slopes is Logarithmic regression. Thank you for your correction, the reference No. was also changed.11. More information is needed to interpret the data.Parallelism: indicate which test was made in order to obtain results of t and pDose response curve: is this a different test or they are results from the parallelism test? Do the equations provide extra information with regard to the parallelism test results (t and p)?Response: Thank you for your suggestion. For the parallelism, a test of equality of two slopes was used to compare between standard slope (C and T) and slope of sample one and sample two obtained from the regression equations. From the equations {dose respone curve) containing value of slope for standard , sample one and two. Based on the results, we compared the standard slope (C and T) with the slope of sample one and sample to see whether they are parallel to standard slope based on the the pvalueand t value. Since the pValue >0.05, it is indicated that these slopes are similar/parallelwith the standard slope (not significant different). More explanation has been added in the revised paper 12. Table 2 and Figure 3 provide the same information. If percentages were used to perform statistics, I suggest removing Table 2.Response: Thank you for your suggestion, the Table 2 has been deleted fronm the revised paper 13. Reviewer 2: used to reliably measure C and T concentrations in serum of Aceh cattleResponse: Thank you, the specific assay used in each study has been added in the revised paper.14. Reviewer 2: Please specify the matrix you are talking aboutResponse: Thank you, the specific assay used in each study has been added in the revised paper.\"The validation of commercial ELISA kits for measuring hormones in animals has been successfully conducted in several animals, such as progesterone in cattle using human progesterone ELISA kit (Clinpro International Co. LLC, Union City, CA 94587, USA) , estradiol-17-beta and inhibin A in buffalo using human ELISA kit (For estradiol: Cat No. EIA-2693, DRG Instruments GmbH, Marburg, Germany; For Inhibin A: Inhibin A DSL-10-28100, Diagnostic System Laboratories Inc, Webster, Texas, US), C in horses using Immunotech kit , and T in Kacang goats using T ELISA kit (Cat No. EIA-1559, DRG Instruments GmbH, Marburg, Germany) developed for human.\" 15. Your results suggest the opposite regarding the T levels Your results suggest the opposite regarding the T levelsResponse: Thank you for your correction, Although, T wes decresed less 10%, but this is not significant. You are right, T is not significantly affected by freeze thaw cycles. The explanation has been improved. 16. Consider shortening the sentence or dividing the information in 2-3 shorter sentences. This way, it will be easier for the reader to understand.Response: Thank you very much for your suggestion. The sentence has been divided into 4 shorter sentences.17. Reviewer 2: Same as before. If the T concentrations remained stable, why do you say that T concentrations decreased?Response: Thank you, it has been corrected in the revised paper.18. Reviewer 2: An important conclusion is lacking: The biological validation suggests that fluctuations of C and T in serum of Aceh cattle have a clear biological significance.Response: Thank you for your suggestion. An additional conclusion has been added in the revised paper.\"In conclusion, our study shows that based on the analytical and biological validation tests, commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring C and T, in serum of Aceh cattle. Moreover, biological validation showed that fluctuations of C and T in serum of Aceh cattle have a clear biological significance reflecting to the physiological condition of Aceh cattle. Results of the study also demonstrate that more than two repeated freeze-thaw cycles significantly affected the stability of serum C concentrations, but up to eight repeated freeze-thaw cycles did not significantly affect the stability of serum T concentrations.\""
}
]
}
] | 1
|
https://f1000research.com/articles/8-1220
|
https://f1000research.com/articles/9-166/v1
|
05 Mar 20
|
{
"type": "Case Report",
"title": "Case Report: Congenital absence of uvula and trismus - a rare presentation of Van der Woude syndrome",
"authors": [
"Victoria Geraldo",
"Abdallah Assaf",
"Muaz Assaf",
"Sohiub Assaf",
"Arshdeep Chauhan",
"Ramzi Ibrahim",
"Victoria Geraldo",
"Abdallah Assaf",
"Muaz Assaf",
"Sohiub Assaf",
"Ramzi Ibrahim"
],
"abstract": "Van der Woude syndrome (VWS) is the most common single gene mutation causing cleft lip/palate, responsible for approximately 2% of all cases. Inherited in an autosomal dominant pattern, VWS occurs at an incidence of 1 in 35,000 to 100,000. The most commonly reported manifestations of VWS is lip pits, cleft lip or palate. We present a case of a 34-week infant with unique and rarely reported symptoms of VWS, such as trismus and absent uvula.",
"keywords": [
"Van der Woude syndrome",
"autosomal dominant",
"gene",
"genetics",
"family history",
"congenital",
"lip pits",
"VWS",
"trismus",
"infant"
],
"content": "Introduction\n\nVan der Woude syndrome (VWS) is the most common single gene cause of cleft lip/palate that has been reported. First described by French Surgeon Jean Nicolas Demarquay in 1845, VWS is a rare autosomal dominant condition with a frequency of 1 in 35,000 to 100,0001–3. The clinical presentation of VWS has been well described in the literature via large multi-generational kindred studies. The most common manifestation and the cardinal feature of VWS is lip pits. This feature is present in approximately 88% affected individuals, and is the only clinical manifestation in 64% of reported cases4. Cleft lip or palate are the only other major hallmark with an estimated occurrence of 20%4. Additional reported signs of VWS are hypodontia, mucous secretion of pits, syngnathia and abnormal brain structure development5–7.\n\nWe present a case of VWS with historically undescribed findings of absent uvula and trismus. We also elaborate on recommendations for managing a newborn that presents with respiratory distress secondary to these pathological findings.\n\n\nCase presentation\n\nA female infant, born at 34-weeks gestation, was delivered via normal spontaneous vaginal delivery to a 20-year-old G3P1011 mother that presented to the Neonatal Intensive Care Unit with worsening respiratory distress. The mother received standard prenatal care and had an unremarkable gestational course. Family history revealed submucosal cleft palate and lip pits in the patient’s mother. Maternal family history was significant for increased incidence of cleft lip/palate, as well as lip pits in multiple relatives (Figure 1). Paternal history/family history were insignificant.\n\nGeneral examination revealed the newborn patient in obvious physical and respiratory distress with copious mucous secretions near the oral orifice (Figure 2). Directed HEENT examination exposed two paramedian indentations with surrounding mound-like elevations exuding mucus located on the lower lip (Figure 3).\n\nVisualization of the oropharynx was limited due to trismus; however, it revealed high arching sub-mucosal cleft palate and absence of a uvula (Figure 4). Directed pulmonary examination revealed substernal as well as intercostal retractions with shallow breathing and sounds indicating upper airway obstruction upon auscultation. The remainder of the physical examination was unremarkable.\n\nHead ultrasound revealed no paramedial structural abnormalities from the high arching and submucosal cleft palate. Chest x-ray revealed mild granularity of lung fields bilaterally slightly more prominent in the right medial lung. Possibility of respiratory distress syndrome or neonatal pneumonia were not excluded.\n\nHospital course was complicated by apneic and desaturation episodes resulting in SpO2 of <88%, requiring intubation and high flow nasal cannula at 4L. Initially thought to be related to prematurity, the poor response and declining respiratory status raised concern and prompted us to investigate further. Aspiration of mucus that was produced within the pitting of the lips seemed to be the culprit to this patient’s respiratory distress. This was further supported by the fact that the patient had an improvement in respiratory status within the decubitus and prone positions versus when the patient was supine.\n\nThe course was further complicated by limitation of oral feeds due to the infant’s inability to adequately open mouth from underlying trismus. This complication was addressed with nasogastric tube placement to aid in food delivery and frequent speech therapy consultations.\n\nThe patient was sent for evaluation by ENT to an outside hospital with three main treatment goals: excision of lip pits to stop mucus secretions and aid in respiratory status, resolution of trismus to aid with initiation of oral feeds and removal of nasogastric tube, and evaluation and repair of submucosal cleft palate.\n\n\nDiscussion\n\nIn an infant presenting with absent uvula or trismus it is important to keep VWS on the differential. It is the largest syndromic cause, making up almost 2% of all cases of orofacial clefts8. Furthermore, when evaluating VWS it is vital to take anticipatory steps in terms of treatment and patient guidance to avoid complications or unwanted outcomes.\n\nIn terms of anticipatory guidance, physicians should emphasize the importance of seeking early orthodontic care and speech therapy to the patient or their caregiver. Seeking orthodontic care and beginning treatment as early as 3 years old has been shown to vastly improve aesthetic outcomes compared to initiating treatment at a later age9. Early speech therapy is a necessary intervention in those with VWS. It has been reported that those with VWS have higher rates of need for speech therapy by age ten than those with non-syndromic cleft lip/palate. It has also been found that those with VWS are twice as likely to undergo procedures to correct their speech problems caused by velopharyngeal dysfunction than those with non-syndromic cleft lip and palate10. With our patient, we initiated inpatient speech therapy consultations to aid with oral feeding, which was imperative in establishing a basis toward patient well-being.\n\nIn terms of treatment, physicians should take early intervention with a multidisciplinary approach. Consultations with appropriate surgical specialties regarding excisions and specifically vertical wedge excisions of lip pits can lead to a good aesthetic outcome as well as avoid complications such as mucocele and pit recurrence11,12. In our case the team felt surgical intervention was necessary to treat and prevent future aspiration-induced respiratory compromise, as seen in the patient.\n\n\nConclusion\n\nVWS can present with an absent uvula and trismus, features that can potentially complicate the hospital course of an infant and impair speech development as the infant ages. Accurate diagnosis of syndromic vs non-syndromic cleft lip and palate can guide treatment. Anticipatory treatment and patient guidance can affect patient outcomes for those with VWS.\n\n\nConsent\n\nWritten informed consent for the publication of the case report including any associated images was obtained from the parents of the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "Acknowledgments\n\nThe authors would like to thank and acknowledge the mother of the child for allowing and trusting us to provide new information to the medical field, while trusting us to keep their personal information private.\n\n\nReferences\n\nDemarquay JN: Quelques considerations sur le bec-de-lievre. Gaz Med Paris. 1845. Reference Source\n\nVan Der Woude A: Fistula labii inferioris congenita and its association with cleft lip and palate. Am J Hum Gene. 1954; 6(2): 244–56. PubMed Abstract | Free Full Text\n\nGorlin RJ, Cohen MM Jr, Hennekam RCM: Orofacial clefting syndromes: common syndromes. In: Motulsky AG Bobrow M Harper PS Scriver C eds. Syndromes of the head and neck. 4th edn. New York: Oxford University Press. 2001. Reference Source\n\nJanku P, Robinow M, Kelly T, et al.: The van der Woude syndrome in a large kindred: variability, penetrance, genetic risks. Am J Med Genet. 1980; 5(2): 117–123. PubMed Abstract | Publisher Full Text\n\nAdvani S, Sogi S, Hugar S, et al.: Vander Woude's syndrome: The rarest of the rare. Contemp Clin Dent. 2012; 3(Suppl 2): S191–S193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPuvabanditsin S, Garrow E, Sitburana O, et al.: Syngnathia and Van der Woude syndrome: a case report and literature review. Cleft Palate Craniofac J. 2003; 40(1): 104–106. PubMed Abstract | Publisher Full Text\n\nNopoulos P, Richman L, Andreasen N, et al.: Abnormal brain structure in adults with Van der Woude syndrome. Clin Genet. 2007; 71(6): 511–517. PubMed Abstract | Publisher Full Text\n\nAngiero F, Farronato D, Ferrante F, et al.: Clinical, histomorphological and therapeutic features of the Van der Woude Syndrome: literature review and presentation of an unusual case. Eur J Paediatr Dent. 2018; 19(1): 70–73. PubMed Abstract | Publisher Full Text\n\nSuda N, Takada J, Ohyama K, et al.: Orthodontic treatment in a patient with Van der Woude's syndrome. Am J Orthod Dentofacial Orthop. 2006; 129(5): 696–705. PubMed Abstract | Publisher Full Text\n\nKitchin S, Grames L, Naidoo SD, et al.: Surgical, Speech, and Audiologic Outcomes in Patients With Orofacial Cleft and Van der Woude Syndrome. J Craniofac Surg. 2019; 30(5): 1484–1487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeralta-Mamani M, Terrero-Pérez Á, Dalben G, et al.: Treatment of lower lip pits in Van der Woude syndrome: a systematic review. Int J Oral Maxillofac Surg. 2018; 47(4): 421–427. PubMed Abstract | Publisher Full Text\n\nSouto, LRM: Congenital Bilateral Lower Lip Pits Associated with Fistulae of the Minor Salivary Glands: Case Report of the Principal Van Der Woude Syndrome’s Trait. Aesth Plast Surg. 2008; 32(1): 172–174. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "62289",
"date": "06 May 2020",
"name": "Emmanuel Adu",
"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\nKey Words Only the following can be considered key words: Van der Woude, congenital lip pits, autosomal dominant, trismus, absent uvula\n\nCase Presentation\nAbbreviations used must be explained e.g. HEENT, G3P1011\n\n“Trismus” is a very important finding yet it was described and also not demonstrated in the picture. Trismus was mentioned because it was limiting visualisation of the oropharynx. No muscle is in spasm, preventing mouth opening. What is being described as trismus is actually syngnatia, a condition caused by fibrous or bony adhesions between the mandible and the maxilla, which is rare finding in patients with Van der Woude syndrome( Ref. No.6).\n\nThe patient has a high arching submucosal cleft palate and an absent uvula. The uvula is absent because the soft palate is also cleft. In a cleft of the soft palate the uvula is absent. It can be recreated only at surgery.\n\nDiscussion\nIn the discussion of the neonate with cleft palate with respiratory obstruction and whose respiratory distress improves when nursed in the prone position, the differential diagnosis should not be limited to Van der Woude syndrome. The Pierre Robin Syndrome (sequence) is the first to be considered. These patients have micrognatia (small chin), glossoptosis, cleft palate and neo-natal respiratory obstruction.\n\nSpeech therapy by age ten is too late. Speech development begins at six months of age. Speech therapy should start by age one\n\nNo mention was made as to the surgical correction of the submucous cleft palate and uvula which is more likely to cause speech problems.\n\nThe title of the paper should read:\n\nVan de Woude Syndrome with syngnathia: a rare presentation\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
},
{
"id": "70441",
"date": "14 Sep 2020",
"name": "Ene-Choo Tan",
"expertise": [
"Reviewer Expertise Human 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\nIntroduction: The data cited in the third and fourth statements in the first paragraph are from a case report of a single kindred published 40 years ago. The frequencies of the specific presentations (which might be specific to this family whose members likely shared the same pathogenic variant) should not be extended to the population of all patients with VWS, unless analysis is done of a large number of cases documented to date.\nFamily history: How was the information obtained? Were the family members examined and confirmed to be affected as recorded in the pedigree? Was it based on medical records? Or as reported by the mother? For those with CL/CP, do/did they have unilateral or bilateral types?\n\nCase presentation: G3P1011 and HEENT should be defined. The 3rd sentence stated “Family history revealed submucosal cleft palate and lip pits in the patient’s mother”. Did the mother say that? Was she examined? No mention was made of surgical treatment carried out and post-surgical outcome, and progress made by the infant at 1-month and 3-months.\n\nDiscussion: Replace the word “largest” in the 2nd sentence “It is the largest syndromic cause…….” Orthodontic care should start before one year for patients with cleft palate, not 3 years old. Is this the first ever finding of absence of uvula and trismus? Does it add anything to the pathway of pattern formation during embryonic development?\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-166
|
https://f1000research.com/articles/9-100/v1
|
10 Feb 20
|
{
"type": "Software Tool Article",
"title": "ccbmlib – a Python package for modeling Tanimoto similarity value distributions",
"authors": [
"Martin Vogt",
"Jürgen Bajorath",
"Martin Vogt"
],
"abstract": "The ccbmlib Python package is a collection of modules for modeling similarity value distributions based on Tanimoto coefficients for fingerprints available in RDKit. It can be used to assess the statistical significance of Tanimoto coefficients and evaluate how molecular similarity is reflected when different fingerprint representations are used. Significance measures derived from p-values allow a quantitative comparison of similarity scores obtained from different fingerprint representations that might have very different value ranges. Furthermore, the package models conditional distributions of similarity coefficients for a given reference compound. The conditional significance score estimates where a test compound would be ranked in a similarity search. The models are based on the statistical analysis of feature distributions and feature correlations of fingerprints of a reference database. The resulting models have been evaluated for 11 RDKit fingerprints, taking a collection of ChEMBL compounds as a reference data set. For most fingerprints, highly accurate models were obtained, with differences of 1% or less for Tanimoto coefficients indicating high similarity.",
"keywords": [
"Bernoulli model",
"fingerprints",
"p-value",
"similarity value distributions",
"Tanimoto coefficient."
],
"content": "Introduction\n\nThe quantitative assessment of molecular similarity is a central concept in chemoinformatics1–4. It forms the basis of similarity searching and ligand-based virtual screening to identify novel molecules in large databases with biological properties similar to given reference compounds5–7. Assessment of molecular similarity plays a central role in chemical space analysis and the study of activity landscapes where chemical space projections onto low-dimensional representations are based on quantified similarities8,9.\n\nThe use of fingerprints and the Tanimoto coefficient10 (Tc), also known as the Jaccard index11, represents one of the most popular methods for quantifying molecular similarity1–4. Fingerprints encode structural features of a molecule in a binary vector format and the Tc quantifies the overlap of features of two molecules as the ratio of the number of common features to the total number of features in each fingerprint. The Tc has the value range 0 to 1 and can be interpreted as the percentage of features shared by two molecules. However, whether a given percentage of overlap should be considered a significant similarity of two molecules depends on the fingerprint design and the global frequency of encoded features. Fingerprint designs might be categorized as dense or sparse. Dense fingerprints have a relatively small dimensionality of at most a few thousand features, but a significant fraction of these might be present in any given molecule. On the other hand, sparse fingerprints can have a theoretically infinite set of features (typical integer encodings allow up to 4 billion features). However, only tens or hundreds of these features might be found in a single molecule. Consequently, sparse fingerprint representations generally lead to smaller Tc values than dense fingerprints.\n\nWhile it is not meaningful to compare Tc values of different fingerprint designs directly, statistical approaches can be applied to assess the significance of Tc values with respect to a reference data set. By using the distribution of Tc values obtained from comparing random compounds as a reference, Tc value significance can be determined by calculating the probability of obtaining a given Tc or higher value by chance. In statistical terms, the reference distribution corresponds to a null hypothesis and the significance measure is known as p-value or p-score. This score has the range 0 to 1 and indicates the probability that a given Tc would be obtained by chance. Thus, smaller p-values indicate higher significance. Here, we will use the measure 1 – (p-value) to assess significance. Although it is in principle possible to obtain Tc distributions by random sampling, this process is time consuming. Instead, the ccbmlib package presented here provides methods for the generation of Tc distribution models that are based on the statistical analysis of feature frequencies and feature correlations between fingerprints for a reference data set. Some mathematical models of Tc-value distributions12–14 have been introduced in the past. The ccbmlib implementation makes use of the conditional correlated Bernoulli model (CCBM) that has been shown to accurately model Tc distributions for a variety of fingerprint designs13,14. An unconditional distribution model accounts for Tc distributions of fingerprints of randomly selected compounds. However, it is of particular interest to model distributions where one compound fingerprint is used as a reference, which forms the basis of similarity searching. P-values obtained from such conditional distribution models efficiently estimate how high a test compound would be ranked in a similarity search with respect to a given reference compound. Hence, conditional models can be used to predict similarity search performance13,14.\n\nThe implementation presented here is based on RDKit15 and provides methods for statistically analyzing fingerprint feature distributions and building models for fingerprints implemented in RDKit. Methods are provided for calculating significance from Tc values, which enable a meaningful comparison of Tc values calculated using fingerprints of different design. The CCBM requires knowledge of the frequency of individual features as well as their pairwise covariances. This statistical analysis needs to be carried out once for each reference data set and fingerprint design. This step can be time consuming for large data sets. The ccbmlib implementation stores resulting statistics permanently to avoid redundant calculations. For our reference implementation and evaluation, compounds from ChEMBL (release 25)16 were selected as a representative sample of bioactive chemical space.\n\n\nMethods\n\nRDKit provides implementations for a variety of fingerprints. Available fingerprints are reported in Table 1. The atom pair fingerprint encodes typed pairs of atoms and their bond distance and is based on the description given by Carhart and Smith17, representing a sparse fingerprint. The Avalon fingerprint18 is a hashed fingerprint enumerating paths and feature classes. MACCS (Molecular ACCess System) keys record the presence or absence of a dictionary of 166 substructural features19. Morgan fingerprints are an RDKit implementation of extended connectivity fingerprints (ECFPs)20 and enumerate atom environments up to a selected radius. We calculated Morgan fingerprints for radius 1 and 2 corresponding to ECFP with diameter 2 and 4, respectively. The topological torsion fingerprints encode sequences of four bonded atoms in a sparse fingerprint21. The RDKit fingerprint is a hashed substructure/path fingerprint similar to the Daylight fingerprints22. Atom pairs, Morgan fingerprints, and the topological torsion fingerprint result in sparse vector representations whose dimensions are only limited by the underlying numerical representation. Hashing is often used to yield a dense fingerprint representation of constant length. We evaluated our models using the sparse and hashed versions with a default size of 2048 bits.\n\nμ(FC) and σ(FC) are the average number and standard deviation of the number of features per fingerprint for ChEMBL compounds, respectively.\n\nFor the following mathematical description of the models, we will use lowercase bold letters to indicate bit vector representations and uppercase italic symbols to denote the corresponding feature set representations:\n\n\n\nHere, d ∈ ℕ is the dimension of the fingerprint.\n\nSimilarity of fingerprints is most often assessed on the basis of the set of features common to two fingerprints. The Tanimoto coefficient10,11 is defined as the ratio of the number of features common to two fingerprints A and B to the total number of features present in either A or B:\n\n\n\nwhere I(A, B) = |A ∩ B| and U(A, B) = |A ∪ B| are the cardinalities of the intersection and union of A and B, respectively.\n\nThe distribution of Tc values depends on the fingerprints of a reference compound data set. The resulting p-values must be interpreted with respect to the reference data set.\n\nAs indicated in Equation 1, fingerprints can be represented as sets of features and similarity metrics like the Tc depend on the cardinalities of the intersection and union of sets. Each of the d features Xi of a fingerprint can be modeled as a Bernoulli variable that occurs with a certain probability pi. Given a reference data set of N compounds and their fingerprints A = {ak|1 ≤ k ≤ N} where ak = (aj1, aj2, … ajd) the probabilities can be estimated from the relative frequencies:\n\n\n\nThe cardinality of a fingerprint itself, of the intersection, and of the union can then be modeled as a sum of non-identically distributed Bernoulli variables. In the case of independent variables, the sum follows a Poisson binomial distribution with mean\n\n\n\nand variance\n\n\n\nand can be approximated by a normal distribution. Because the cardinalities of the intersection and union of two sets are not independent, the Tc is then modeled as the ratio of two correlated normal distributions for which approximations exist23,24.\n\nFingerprint features are often correlated. Ignoring these correlations leads to a significant underestimation of the variance (Equation 5)13,14. While the equation for the mean μ remains valid for correlated random variables, the formula for the variance σ2 requires taking the pairwise covariances cij = cov(Xi,Xj) between the different features into account. These can also be estimated from the reference set:\n\n\n\nAccordingly, the value cii = pi (1 – pi) denotes the variance of Xi.\n\nBased on these estimates, the average cardinality of a fingerprint itself, of the intersection, and of the union of two unknown fingerprints can be determined:\n\n\n\n\n\n\n\nFor the respective variances, one obtains:\n\n\n\n\n\n\n\nThe covariance between the cardinality of union and intersection is given by:\n\n\n\nNormal distributions are defined by their mean and standard deviation and can thus be calculated from the estimates of the averages and variances. However, given the fact that the underlying features are not independent, the suitability of using normal distributions as approximations cannot be guaranteed from a theoretical point of view. Nevertheless, as has been previously shown13,14, and as can be seen from our current evaluation (vide infra), practical applications of the model yield good performance for a variety of different fingerprint designs. Under the assumption of normality, the following models are obtained:\n\n\n\n\n\nwhere N(μ,σ2) is the normal distribution with mean μ and standard deviation σ. The Tc distribution is then modeled as a ratio of these two correlated distributions. An analytical form of the probability distribution function exists23; however, for determining p-values and the significance, the following approximation of the cumulative distribution function (CDF) is used24:\n\n\n\nHere, ρ = covIU / (σIσU) is the correlation between intersection and union and Φ is the CDF of the standard normal distribution:\n\n\n\nThe p-value can then be determined as:\n\n\n\nFor model evaluation, we use F(t) = Pr (Tc ≤ t) directly as an indication of significance.\n\nFor similarity searching, reference compounds are used and Tc values of database compounds are calculated relative to the references. As has been shown13, distributions of Tc values can vary greatly depending on the reference fingerprint. In this case, the significance of Tc values should to be considered for a given reference compound. Mathematically, this corresponds to determining the conditional distributions when one fingerprint is given. As in the unconditional case, the distributions are based on sums of correlated Bernoulli variables that are modeled as normal distributions based on the conditional means and variances:\n\n\n\n\n\n\n\n\n\n\n\nThe conditional model is obtained by applying these parameters in Equation 16.\n\nA derivation of the formulas presented here for the CCBM can be found in the original publications13,14.\n\nSparse fingerprints like ECFPs or the Morgan fingerprint might result in hundreds of thousands of different features present in large data sets. Most of these will occur with very small probabilities pi and only have a small influence on the estimated means and variances. It is computationally unproblematic to handle these individual probability estimates; however, determining pairwise covariances of all possible features becomes infeasible for more than a few thousand features. To address this issue, the complete covariance matrix is only determined for the most frequent features of a sparse fingerprint (by default, the 2048 most frequent features are selected). Covariances involving rare fingerprints are not estimated. Given that feature probabilities of combinatorial fingerprints usually show pseudo-exponential drop-offs for rare features, contributions towards covariance estimates have negligible influence on the final estimates and are ignored in the current implementation.\n\nAs reference data set, ChEMBL compounds were selected. SMILES representations of 1,870,461 compounds were downloaded and standardized using a previously published protocol included in the ccbmlib package25. Additionally, stereochemical information was removed since most fingerprints implemented in RDKit do not account for stereochemistry, resulting in 1,691,786 unique compounds. Fingerprint statistics are reported in Table 1.\n\nThe software has been implemented as a module for Python 3.7. It requires the installation of RDKit and has been tested with version 2019.03.4 of RDKit. Any system (Linux, Windows, MacOS) capable of running Python 3.7 and RDKit is sufficient for running our software. A 64-bit operating system with at least 8GB RAM is recommended. After obtaining the code it can be installed using Python’s setup utility. The ccbmlib package contains three modules: preprocessing, statistics, and models.\n\nModule preprocessing consists of routines for standardizing molecules and preparing compound data sets. Standardization of molecules is a generally recommended preprocessing step, especially when compound data sets are assembled from different sources.\n\nModule statistics contains classes for feature statistics and distribution models. Its main classes are PairwiseStats and CorrelatedNormalDistributions for the fingerprint statistics and distribution models, respectively. Distribution models are obtained from PairwiseStats objects using the get_tc_distribution method, which are used to generate unconditional and conditional models.\n\nThe module models provides the main interface for the package. It offers wrapper functions for calculating RDKit fingerprints and contains the central method get_feature_statistics for generating or retrieving fingerprint statistics for a reference data set. Once calculated, statistics are saved and can be retrieved for later use. Exemplary applications of the module are provided in the readme file of the ccbmlib distribution.\n\n\nResults and discussion\n\nFingerprint statistics were calculated on the basis of the 1,691,786 unique ChEMBL compounds and distribution models were derived. To evaluate the quality of the general model, 1,000,000 Tc values were calculated from pairs of random compounds drawn from the ChEMBL data set and empirical CDFs were determined. Figure 1 compares the empirical CDFs to the modeled unconditional CDFs for the fingerprints in Table 1. Overall, the modeled CDFs match the different value ranges and shapes of the empirical CDFs very well. However, to assess the usefulness of the model as a quantitative and comparative tool, the quality of the model should be assessed with a focus on Tc values indicating high significance. The insets of the figures show an enlarged section with Tc values having a significance of 0.9 or higher. The models for the atom pair fingerprints are not able to accurately model the distribution in this region. However, most other Tc distributions can be modeled very well. For the MACCS, Morgan, and topological torsion fingerprint distributions, high-quality models are obtained with small differences between the theoretical and empirical model. The hashed variants of the Morgan and topological torsion fingerprints have distributions highly similar to their sparse counterparts. This can be expected because the average feature counts reported in Table 1 are also very similar, indicating that most of the sparse features are hashed to unique values and only few collisions occur between hashed values. The path-based Avalon and RDKit fingerprints still have usable, although less accurate models. These observations are consistent with previous observations13. CCBM models pharmacophore-based fingerprints only to a limited extent. This might be due to the specific nature of correlations between pharmacophore features.\n\nThe empirical and modeled cumulative distribution functions for the fingerprints reported in Table 1 are shown in (a) – (k). Blue lines indicate empirical distributions obtained from randomly sampling 1,000,000 pairs of compounds from ChEMBL. Red lines show the corresponding modeled distributions according to Equation (16). The inserts highlight the correspondence between the curves for Tc values of high significance.\n\nA quantitative summary of the observations is given in Table 2. It reports the Kolmogorov-Smirnov statistic (KS)26, which is defined as the maximum difference between empirical (Femp) and modeled (Fmodel) distributions:\n\n\n\nKS reports the Kolmogorov-Smirnov statistic comparing the experimental to the modeled distributions. KS90 reports the Kolmogorov-Smirnov statistic limited to Tc values with an empirical significance of at least 90%.\n\nIn addition, the maximum difference for the significance range beyond 90% is reported (KS90):\n\n\n\nThe maximum difference for most models is observed for common Tc values, i.e., where the slope of the CDF is steepest. However, as can be seen from the KS90 values, the high significance range can be accurately assessed within 1% for MACCS, most Morgan, the torsion, and the Avalon fingerprints. The RDKit fingerprint still performs reasonably well with a KS90 of 1.70, whereas values of 4.22 and 8.80 for the atom pair fingerprint and its hashed variant indicate poor performance of the model in this region.\n\nIn addition to the unconditional model, conditional distributions were investigated when a reference fingerprint was given. As each reference fingerprint will yield a different model, 100 compounds were randomly chosen as a reference and conditional models were derived and compared to empirical Tc distributions by comparing the reference compound to 100,000 randomly chosen compounds. The ranges of correspondences between empirical and modeled significance values are shown in Figure 2. The MACCS and Morgan fingerprints again showed the best conditional models, all of which were close to the ideal diagonal. For most reference compounds, the topological torsion fingerprint also yielded very good models; however, few outliers with large deviations were observed. This might be expected when reference fingerprints only contain very few features and approximations by normal distributions fail to yield accurate models.\n\nFor the fingerprints in Table 1, each of the graphs (a) – (k) shows the variation of correspondences between empirical and modeled significance values of 100 conditional distributions obtained by selecting random reference compounds. Empirical distributions for each reference compound were determined from comparisons of 100,000 randomly chosen compounds. The black line indicates the median correspondence between empirical and modeled distribution. The dark gray area shows the interquartile range and the light gray area the range from the 5th to the 95th percentile. The green line is the diagonal corresponding to a perfectly matching model. The inserts highlight correspondences for significance values larger than 0.9.\n\nThe Python code used for data generation, data analysis, and generation of the figures is available in form of a Jupyter notebook in the github repository27.\n\n\nConclusions\n\nThe tools provided make it possible to evaluate the significance of Tc values for a variety of fingerprints from RDKit. Users can generate distribution models for different fingerprints with respect to reference data sets. Accurate models are obtained for most RDKIT fingerprints including the popular MACCS and Morgan fingerprints. Based on these models, it can be assessed to what extent molecular similarity is accounted for by fingerprints of different design and to what extent similarity between compounds sharing the same activity is reflected by similarity scores calculated on the basis of different fingerprint representations. Furthermore, the conditional models can be used to predict the suitability of fingerprints for similarity searching and ligand-based virtual screening.\n\n\nData availability\n\nThe data sets used in this paper are freely available from ChEMBL: https://www.ebi.ac.uk/chembl/\n\nSmiles structure representations were retrieved on 15 Jan 2020 from: ftp://ftp.ebi.ac.uk/pub/databases/chembl/ChEMBLdb/latest/chembl_25_chemreps.txt.gz\n\n\nSoftware availability\n\nOur package depends on RDKit, which is freely available from https://www.rdkit.org\n\nSource code is available from: https://github.com/vogt-m/ccbmlib\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.363495327\n\nLicense: MIT",
"appendix": "References\n\nWillett P, Barnard JM, Downs GM: Chemical similarity searching. J Chem Inf Comp Sci. 1998; 38(6): 983–996. Publisher Full Text\n\nWillett P: Similarity methods in chemoinformatics. Ann Rev Inf Sci Technol. 2009; 43(1): 1–117. Publisher Full Text\n\nMaggiora GM, Shanmugasundaram V: Molecular similarity measures. In Chemoinformatics and computational chemical biology. Humana Press, Totowa, NJ. Methods Mol Biol. 2011; 672: 39–100. PubMed Abstract | Publisher Full Text\n\nMaggiora G, Vogt M, Stumpfe D, et al.: Molecular similarity in medicinal chemistry: miniperspective. J Med Chem. 2014; 57(8): 3186–3204. PubMed Abstract | Publisher Full Text\n\nEckert H, Bajorath J: Molecular similarity analysis in virtual screening: foundations, limitations and novel approaches. Drug Discov Today. 2007; 12(5–6): 225–233. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Similarity searching. Wiley Interdiscip Rev Comput Mol Sci. 2011; 1(2): 260–282. Publisher Full Text\n\nWillett P: Combination of similarity rankings using data fusion. J Chem Inf Model. 2013; 53(1): 1–10. PubMed Abstract | Publisher Full Text\n\nMaggiora GM, Bajorath J: Chemical space networks: a powerful new paradigm for the description of chemical space. J Comput Aided Mol Des. 2014; 28(8): 795–802. PubMed Abstract | Publisher Full Text\n\nGuha R: Exploring structure–activity data using the landscape paradigm. Wiley Interdiscip Rev Comput Mol Sci. 2012; 2(6): 829–841. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers DJ, Tanimoto TT: A computer program for classifying plants. Science. 1960; 132(3434): 1115–1118. PubMed Abstract | Publisher Full Text\n\nJaccard P: The distribution of the flora in the alpine zone. New phytol. 1912; 11(2): 37–50. Publisher Full Text\n\nBaldi P, Nasr R: When is chemical similarity significant? The statistical distribution of chemical similarity scores and its extreme values. J Chem Inf Model. 2010; 50(7): 1205–1222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVogt M, Bajorath J: Introduction of the conditional correlated Bernoulli model of similarity value distributions and its application to the prospective prediction of fingerprint search performance. J Chem Inf Model. 2011; 51(10): 2496–2506. PubMed Abstract | Publisher Full Text\n\nVogt M, Bajorath J: Modeling Tanimoto Similarity Value Distributions and Predicting Search Results. Mol Inform. 2017; 36(7): 1600131. PubMed Abstract | Publisher Full Text\n\nRDKit: open-source cheminformatics software. (accessed Jan 27, 2020). Reference Source\n\nGaulton A, Hersey A, Nowotka M, et al.: The ChEMBL database in 2017. Nucleic Acids Res. 2017; 45(D1): D945–D954. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarhart RE, Smith DH, Venkataraghavan R: Atom pairs as molecular features in structure-activity studies: definition and applications. J Chem Inf Comp Sci. 1985; 25(2): 64–73. Publisher Full Text\n\nGedeck P, Rohde B, Bartels C: QSAR--how good is it in practice? Comparison of descriptor sets on an unbiased cross section of corporate data sets. J Chem Inf Model. 2006; 46(5): 1924–1936. PubMed Abstract | Publisher Full Text\n\nMACCS Structural Keys. Accelrys: San Diego, CA. 2011. Reference Source\n\nRogers D, Hahn M: Extended-connectivity fingerprints. J Chem Inf Model. 2010; 50(5): 742–54. PubMed Abstract | Publisher Full Text\n\nNilakantan R, Bauman N, Dixon JS, et al.: Topological torsion: a new molecular descriptor for SAR applications. Comparison with other descriptors. J Chem Inf Comp Sci. 1987; 27(2): 82–85. Publisher Full Text\n\nDaylight Theory manual. Daylight Chemical Information Systems, Inc : Laguna Niguel, CA. (accessed Jan 27, 2020). Reference Source\n\nMarsaglia G: Ratios of normal variables and ratios of sums of uniform variables. J Am Stat Assoc. 1965; 60(309): 193–204. Publisher Full Text\n\nHinkley DV: On the ratio of two correlated normal random variables. Biometrika. 1969; 56(3): 635–639. Publisher Full Text\n\nde la Vega de León A, Lounkine E, Vogt M, et al.: Design of diverse and focused compound libraries. In: Tutorials in Chemoinformatics. John Wiley & Sons Ltd, Chichester, UK. 2017; 83–101. Publisher Full Text\n\nBirnbaum ZW, Tingey FH: One-Sided Confidence Contours for Probability Distribution Functions. Ann Math Stat. 1951; 22(4): 592–596. Reference Source\n\nVogt M, Bajorath J: ccbmlib – a Python Package for Modeling Tanimoto Coefficient Distributions for Molecular Fingerprints (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3634953"
}
|
[
{
"id": "59806",
"date": "28 Feb 2020",
"name": "Brian Goldman",
"expertise": [
"Reviewer Expertise machine learning for computational chemistry",
"statistics."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article ‘ccbmlib: a Python package for modeling Tanimoto similarity value distributions’, by Vogt and Bajorath is clearly written and concretely describes a method for determining the significance of tanimoto similarity scores. The statistical technique detailed in the paper outlines a mathematical method for converting tanimoto similarity scores from various binary molecular fingerprints into significance (p) values. Consequently, the method provides a way of normalizing similarity scores so that comparisons between results of searches utilizing different fingerprinting methods can be conducted easily. The paper also outlines a ‘conditional method’ that provides a technique for estimating the distributions of similarity scores for a given reference compound. This allows one to estimate how well a test compound would rank in a large-scale similarity search.\n\nThe explanations and mathematical equations in the paper are easy to follow. The graphs in the results section clearly support the findings of the study. I would recommend this paper to be indexed in its current form.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5270",
"date": "05 Mar 2020",
"name": "Jürgen Bajorath",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Thank you for your instructive comments on the manuscript."
}
]
},
{
"id": "59805",
"date": "28 Feb 2020",
"name": "David A. Cosgrove",
"expertise": [
"Reviewer Expertise Cheminformatics software development within the pharmaceutical industry."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a method for analysing the occurrence of features in a set of fingerprints that have been generated from a reference collection of chemical structures. They use this analysis to generate models for assessing the statistical significance of the tanimoto coefficients for pairs of fingerprints in the set. Using the model, they can produce a plot of significance vs tanimoto coefficient (a CDF). In the paper, the accuracy of the model is assessed by comparing the curve so produced with those created by calculating the tanimoto coefficients for pairs of fingerprints from a large random sample of the set. The correspondence between the modelled and empirical distribution functions is high.\nThe paper is clearly laid out and relatively easy to read, if one takes the maths at face value. It is likely that it would be possible to reproduce their analysis from the information given. However, that is not strictly necessary from a practical standpoint as the authors have made the software they have developed for the analysis available as a Python module for anyone to download and use. They are to be commended for this action, which is still rare in the field of cheminformatics. It is likely to increase the impact of the paper considerably.\nWhen I read a paper of this nature, a key question I pose myself is “how, if at all, will this help me with my work?” Here I fear the authors have been less successful. For example, there is an implementation in the RDKit toolkit of the Taylor-Buttina clustering method. This is a popular way of clustering fingerprints, and hence molecules, that is widely used for things like analysis of high-throughput screening results, organising the results from a virtual screen etc. A key input parameter to the algorithm is a threshold tanimoto coefficient – all fingerprints within a cluster are guaranteed to be within this similarity of the first fingerprint placed in the cluster. The success of this method for clustering depends very strongly on the value chosen for this threshold. Too high, and one obtains an unhelpfully large number of small clusters; too low, and the clusters will be large and contain molecules without apparent similarity. It would be very useful if there were a way of taking a successful threshold for one fingerprint type and using it to decide upon a similarly successful threshold for a different type. I feel as though this paper contains a way of doing this, but it is unclear to me quite how it would be achieved with the results presented. If the authors could add to the paper an example of how one would take a CDF for one fingerprint type and use it to translate a useful tanimoto coefficient threshold for it into an equally useful threshold for a different fingerprint type, that would, in my opinion, make the paper much more valuable.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5269",
"date": "05 Mar 2020",
"name": "Jürgen Bajorath",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Thank you for your comments and your suggestion. Indeed, a potential application of the methodology is establishing correspondences between Tc values of different fingerprints according to their statistical significance. Therefore, a paragraph has been added to the manuscript explaining how modeled distributions can be used to identify corresponding Tanimoto coefficients (Tc values) for fingerprints of different design. In addition, a figure has been added displaying the relationship between MACCS Tc values and Tc values of other fingerprints. The software and Jupyter notebook have been updated accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/9-100
|
https://f1000research.com/articles/7-1523/v1
|
21 Sep 18
|
{
"type": "Research Note",
"title": "Root mass may affect soil water infiltration more strongly than the incorporated residue",
"authors": [
"Masato Oda",
"Burhanuddin Rasyid",
"Hide Omae",
"Burhanuddin Rasyid",
"Hide Omae"
],
"abstract": "Crop residue incorporation increases stable soil pores and soil water infiltration and reduces surface water runoff and soil erosion. However, few studies have examined the relationship between crop residue incorporation and water infiltration. A previous study showed that water infiltration increases depending on the quantity of applied wheat straw. In this study, we examined whether the relationship is applicable to different crop residues in a crop rotation. We grew corn, rose grass, and okra in crop rotation under plastic film houses and measured the water infiltration rate at the time of ridge making. A strong correlation was found between the quantity of applied residue and the soil water infiltration rate (r = 0.953), although there are outliers in the case of no prior crop. However, aboveground biomass of the prior crop showed a stronger correlation with water infiltration rate (r = 0.965), without outliers. Previous studies have revealed the exponential relation between plant root mass and soil erosion. Our data also show a positive relationship between resistance to erosion and root mass when assuming that aboveground biomass is proportional to the underground biomass. The result also showed that the effect of the prior crop root mass disappears within the next crop period. Our results indicate that maintaining a large root biomass is crucial for reducing soil erosion.",
"keywords": [
"Crop residue",
"Crop rotation",
"Soil erosion",
"Soil water infiltration",
"Sustainable agriculture"
],
"content": "Introduction\n\nSoil degradation is a major constraint of food security (Gomiero, 2016; Lal, 2015), and soil erosion represents one of the crucial intervention points for reversing soil degradation (Karlen & Rice, 2015). The no-tillage method is a major approach to tackle erosion; however, tillage remains a major management approach on arable land. The Universal Soil Loss Equation (USLE) (Wischmeier & Smith, 1978) is the standard for estimating erosion. The equation shows that the risk of erosion is reduced when a crop has covered soil surface. This emphasizes the importance of preventing erosion in the early stage of crop growth. In general, it is rare for rain intensity to exceed 30 mm 10 min−1 (Kouhei et al., 2011). Therefore, surface runoff after tillage almost never occurs if the infiltration rate of a field is larger than 30 mm 10 min−1. Therefore, technologies increasing the infiltration rate higher than the precipitation rate are needed to prevent soil erosion in tillage systems.\n\nCrop residue management is a crucial factor in the early stage of crop growth. Tillage makes soil porous, but the physical properties are rapidly lost (Strudley et al., 2008). However, organic matter application increases the stability of soil pores (Turmel et al., 2015). Potter et al. (1995) reported that water infiltration of soil was higher under no-tillage than tillage conditions when the residue input was low, but the opposite result was shown when the residue input was high. The soil erosion decreased according to the degree of water infiltration (Potter et al., 1995). Just after residue incorporation, rill erosion rates were significantly reduced (Brown et al., 1989). A previous study reported that surface water runoff under normal subtillage reduced up to the applied wheat straw quantity, as the water infiltration increased with the quantity of applied straw (Russel, 1940), although the relation between the quantity of applied residue and infiltration rate has been less studied. Conversely, it is known that plant root mass is related to rill and ephemeral gully erosion (Gyssels et al., 2005).\n\nOur research aims were as follows: 1) to determine whether the relation between residue incorporation and infiltration holds under crop rotation, and 2) to determine whether the remaining underground root mass influences this relation. To address these questions, we designed an experiment that involved ensuring growth of different crops for the same amount of input residue with different nutrition levels and different soil moisture levels.\n\n\nMethods\n\nWe conducted the experiment in two plastic film houses at the Japan International Research Center for Agricultural Sciences experimental field (24.38°N and 124.19°E) on Ishigaki Island. The climate is subtropical. The soil type was Ultisol (Soil Survey Staff, 2014) and the texture was sandy clay loam. The house was 5 m wide and 18 m long. We made three soil ridges (0.2 m high and 1 m wide) with a 0.5 m path on each side. We divided these ridges into three plots with 0.8 m paths between each plot. In this way, we created nine plots (1 m × 5.2 m) in each film house and randomly assigned them with nine treatments (3 × 3 factorial design). These treatments comprised three nitrogen levels (0, 10, and 40 kg N ha−1; slow-release-type urea only, no other fertilizers were used) and three soil moisture levels (unmulched, weed barrier fabric, and black plastic film mulch).\n\nThe effect of the soil moisture difference treatment was determined at the end of okra cropping by extracting soil core samples from 0 to 5 cm soil depth on the ridge. We replicated the treatments using two film houses (A and B). We cropped corn (Zea mays) without fertilizer before the experiment and collected the residue, then chopped the residue into approximately 3 cm pieces using a chopper and dried it for a month under a roof. We adjusted the soil moisture of the house at a suitable level for tillage by irrigating (25–40 mm) with mist irrigation tubes (Kiriko; Mitsubishi Chemical Agri Dream Co., Ltd., Tokyo) and then removed the tubes. We scattered 2 Mg ha−1 of the corn residue, tilled by a rotary tiller, made the ridges, measured the soil water infiltration, set the irrigation tubes again, set the mulch films, transplanted rose grass (Chloris gayana) seedlings with fertilizer, and irrigated up to the field capacity. Additional irrigation was not provided. After harvesting rose grass, we repeated the above processes in the same way for okra (Abelmoschus esculentus). All the crop residues were collected in each house then evenly returned to the plots (each plot received the same amount of residue for the next crop per house; the amount was different between the houses). The growing season of corn, rose grass, and okra were 7 June to 10 August 2016, 14 October 2016 to 11 January 2017, and 12 January to 14 April 2017, respectively. An interval of 65 days was provided between the corn harvesting and the rose grass planting. There was no interval between rose grass harvesting and okra planting.\n\nWe measured the soil water infiltration rate with Mariotte's bottle (20 cm high, 10 cm in diameter), with two holes in the bottom. Mariotte’s bottle is a device that delivers a constant rate of flow. We inserted a plastic ring of the same diameter into the ridge to a 10 cm depth and then watered from a 1 m height to the ring at a 60 mm min−1 rate. We recorded the time needed to waterlog 50% of the soil surface area. We measured infiltration on the ridge at the initial stage (before the rose grass; with incorporated corn residue), after the rose grass (with incorporated rose grass residue), and after the okra (with incorporated okra residue).\n\nAboveground biomass was calculated by multiplying the plot’s whole fresh biomass weight to the average moisture content of the air-dried samples’ in each house. We performed Pearson’s product moment correlation analysis of the infiltration rate for the quantity of applied residue or for the aboveground biomass (dry weight) using the “CORREL” function of MS Excel 2016. The correlation coefficients were calculated for the mean values of nitrogen levels and for that of the mulch levels. The mean values of nitrogen levels show the effects of aboveground biomass, which averaged out the effect of soil moisture. By contrast, the mean values of mulch levels show the effect of soil moisture.\n\n\nResults\n\nThe incorporated residues of each house were 2.0 and 2.0 Mg ha−1 for the initial stage (before seeding the rose grass), 3.8 and 4.7 Mg ha−1 after the rose grass, and 0.8 and 0.8 Mg ha−1 after the okra. The corresponding infiltration rates were 45 and 36 mm, 97 and 123 mm, and 32 and 47 mm, respectively. There was a strong correlation between the quantity of incorporated residue dry weight and soil water infiltration rate (r = 0.953) in terms of nitrogen level treatment, although initial corn residue showed outliers (Figure 1a). However, aboveground biomass of the prior crop showed a higher correlation with soil water infiltration rate (r = 0.965), without outliers (Figure 1b). The correlation coefficient of the infiltration rate and the aboveground weight decreased to r = 0.872 for the mulch level treatment (Figure 1c). The soil moisture (0–5 cm) of the N0, N1, and N4 treatment at the end of okra cropping was 8.3% (±1.6%), 7.2% (±1.0%), and 7.8% (±1.7%), respectively (Oda et al., 2018). The soil moisture of the unmulched, the fabric, and the film treatments were 6.5% (±0.7%), 7.6% (±0.8%), and 9.7% (±0.9%), respectively (Oda et al., 2018).\n\n(a, b) Means of the nitrogen-level treatment. (c) Means of the mulch-level treatment. Crop rotation was conducted as follows: corn, rose grass, and okra in plastic film houses. An interval of 65 days was provided between the corn harvesting and the rose grass planting. There was no interval between rose grass harvesting and okra planting. Houses A and B are replicates. We measured the soil infiltration rates on the ridge using artificial rainfall equipment on the day of making the ridge. The values are the mean of three plots.\n\n\nDiscussion and conclusions\n\nWe found a strong correlation between input residue and the infiltration rates for the nitrogen-level treatment (Figure 1a). However, the average infiltration rate of initial stage was almost the same as that of after okra, although the input quantity of the initial stage (2.0 Mg ha−1) was a 2.5-fold higher than after okra (0.8 Mg ha−1). By contrast, the correlation for the aboveground biomass had no outliers (Figure 1b). This reveals that aboveground biomass affects infiltration rate more than the applied residue. A previous study has shown that the decrease in water erosion rates with increasing root mass is exponential, although infiltration was not mentioned (Gyssels et al., 2005). Our data show a positive relationship between resistance to erosion and root mass when assuming that aboveground biomass is proportional to the underground biomass.\n\nThe mulch treatment data shows that correlation between the aboveground weight and infiltration rate was erratic (Figure 1c). The soil moisture difference caused large differences in aboveground growth. In addition, the unit performance of infiltration was not stable. The difference of soil water content at most 3.2% (6.5% of the unmulched and 9.7% of the film) in top 5 cm was negligible against the 25–40 mm of applied water before measurement for making the ridge. Therefore, it is considered that the outliers did not come from the difference of soil moisture but came from the difference of root mass, which was affected by the soil dryness.\n\nWe should consider the duration of the “after-effect” of the prior crop, such as the roots of rose grass on the soil water infiltration rate measurement of after okra (Wischmeier & Smith, 1978). Two facts support the observation of a small after-effect. Firstly, the infiltration rates were very small (32 and 47 mm) after okra, although they were very large (97 and 123 mm) after rose grass. Secondly, the correlation between the aboveground biomass and the infiltration rate was stable and less was affected by a prior crop. Therefore, we conclude that the effect of the prior crop root mass almost disappears within the next crop growth period under the experimental conditions.\n\nFinally, the key finding of this study is that the effect of aboveground residue quantity, more precisely root mass, was stronger than the incorporated residue. Although the residue quantity was the main factor determining the infiltration rates at tillage, the fairly low performance of initial corn residue is remarkable. This is likely to be because interval between harvesting corn and rose grass planting was very high (65 days). The soil pores would have been decreased. From a physical viewpoint, the area of residue surface is far smaller than that of the root surface. And the gap is easily clogged by sediment caused by rainfall. Therefore, the improvement of soil water infiltration probably comes from root mass (Gyssels et al., 2005). This suggests the importance of sequential cropping in tropical regions.\n\n\nData availability\n\nRaw data of this article are presented in figshare: https://doi.org/10.6084/m9.figshare.6741890.v1 (Oda et al., 2018).\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 thank Masato Shimajiri, Yasuteru Shikina, Masashi Takahashi and Masahide Maetsu for their assistance with experiment. We thank Akiko Sawa for her assistance with the sample analysis.\n\n\nReferences\n\nBrown LC, Foster GR, Beasley DB: Rill Erosion as Affected by Incorporated Crop Residue and Seasonal Consolidation. Trans ASAE. 1989; 32(6): 1967–1978. Publisher Full Text\n\nGomiero T: Soil Degradation, Land Scarcity and Food Security: Reviewing a Complex Challenge. Sustainability. 2016; 8(3): 281. Publisher Full Text\n\nGyssels G, Poesen J, Bochet E, et al.: Impact of plant roots on the resistance of soils to erosion by water: a review. Prog Phys Geogr. 2005; 29(2): 189–217. Publisher Full Text\n\nKarlen DL, Rice CW: Soil Degradation: Will Humankind Ever Learn? Sustainability. 2015; 7(9): 12490–12501. Publisher Full Text\n\nKouhei A, Noriyuki Y, Kiyoshi O, et al.: A-03.pdf. In: A consideration on secular change of rainfall characteristics in Okinawa prefecture.2011.\n\nLal R: Restoring Soil Quality to Mitigate Soil Degradation. Sustainability. 2015; 7(5): 5875–5895. Publisher Full Text\n\nOda M, Rasyid B, Omae H: Crop residue and soil water infiltration. figshare. Dataset. 2018. http://www.doi.org/10.6084/m9.figshare.6741890.v1\n\nPotter KN, Torbert HA, Morrison JE Jr: Tillage and residue effects on infiltration and sediment losses on Vertisols. Trans ASAE. 1995; 38(5): 1413–1419. Publisher Full Text\n\nRussel JC: The Effect of Surface Cover on Soil Moisture Losses by Evaporation1. Soil Sci Soc Am J. 1940; 4: 65. Publisher Full Text\n\nStrudley MW, Green TR, Ascough JC: Tillage effects on soil hydraulic properties in space and time: State of the science. Soil Tillage Res. 2008; 99(1): 4–48. Publisher Full Text\n\nTurmel MS, Speratti A, Baudron F, et al.: Crop residue management and soil health: A systems analysis. Agric Syst. 2015; 134: 6–16. Publisher Full Text\n\nWischmeier WH, Smith DD: Predicting rainfall erosion losses. USDA, Maryland. 1978. Reference Source"
}
|
[
{
"id": "43591",
"date": "07 Feb 2019",
"name": "Kae Miyazawa",
"expertise": [
"Reviewer Expertise soil science",
"vegetable cultivation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 short report to investigate the infiltration rate under different treatments, conducted in 2 green houses. The treatments were 3 levels of urea input, and 3 ways for mulching. They have grown corn, rose grass, and okra, and incorporated them after each cropping season, and measured infiltration rates. Their conclusion is that the root mass affects infiltration more than incorporated residue amount, but it's flawed (details below).\n\nIntroduction:\nThe introduction has to be revised. Right now, two paragraphs that are supposed to give the background for your research aim are not structured well. Right now, you have just put related information without considering logical order. It can be improved, for example, if the first paragraph is structured like this;\n1. Erosion is a major problem for the soil degradation. 2. Erosion starts when rain hits the bare soil and form a crust by braking aggregates, which decreases infiltration and increase surface runoff. 3. Therefore, to reduce soil erosion, protecting soil surface and improving infiltration rate are important.\n\nThen, in the second paragraph, you can talk about how to do that (no tillage, putting residues, incorporating organic matter etc.). Also at the end, or the next paragraph, you can talk about what is still lacking and needs to be investigated to show the originality of this report.\n\nIn the last paragraph of the introduction, you put your research aim. 1), and 2) are understandable, but the last sentence is not clear. Why can \"ensuring growth of different crops for the same amount of input residue with different nutrition levels and different soil moisture levels\" provide answer for those two questions?\n\nMethods:\nYou have N fertilizer treatment, and \"soil moisture\" treatment, but putting as \"three soil moisture levels (un-mulched, ....)\" is not really exact naming for your treatment, since you are not really controlling soil moisture alone (covering with mulch has a lot more effect other than soil moisture). Probably \"mulching treatment\"?\n\nSecond paragraph: Please put the information in order. You are talking about the end of Okura cropping, then going back to the corn cropping next. Please make it easy for readers to understand by putting them in logical manner.\n\n\"we repeated the above processes\": What are the processes? There are so many processes in \"above\". Do you mean all including corn cropping?\n\nIt is not clear whether you returned residues evenly among plots within the house, or across the houses.\n\nData analysis: Since this experimental design is 2 factor, 3 levels, randomized block design with 2 replication (block), please conduct 2-way anova accordingly.\n\nResults, Discussion and conclusions:\nIf you use the 2-way ANOVA results, you might find something interesting. Since the residue amount increases as the crop biomass and crop root biomass increases, these results are basically showing the same thing. When you had more residue (for example, when you have grown rose grass), you also had more above ground and root biomass. So the Figure 1a result is concomitant with the crop biomass (and root biomass), and not purely showing the effect of the residue amount. If you want to compare the residue incorporation effect and crop biomass (and root biomass), you need to have plots with uniform root biomass condition, and incorporate residues in different levels.\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? No\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": "4441",
"date": "28 Feb 2019",
"name": "Masato Oda",
"role": "Author Response",
"response": "First of all, we respect you for bravely participating in the open review. As you know, the open review has not been popular in our field of sciences. We are deeply thankful to you. And it is our pleasure to enjoy the discussion with you. We are also thinking that your comments are on behalf of many other readers. We understand that the discussion presents them with a correct understanding of our results by improving the manuscript.Comment 1:Introduction:The introduction has to be revised. Right now, two paragraphs that are supposed to give the background for your research aim are not structured well. Right now, you have just put related information without considering logical order. It can be improved, for example, if the first paragraph is structured like this;1. Erosion is a major problem for the soil degradation.2. Erosion starts when rain hits the bare soil and form a crust by braking aggregates, which decreases infiltration and increase surface runoff.3. Therefore, to reduce soil erosion, protecting soil surface and improving infiltration rate are important.MO: Yes, you are right. We need to revise the introduction. However, the order is different. We showed the importance of the infiltration rate from the holistic view of preventing soil erosion practically. For that purpose, we are referring USLE. From this point of view, we showed that erosion is a problem of mainly combined with tillage. We added a sentence as follows: “What is the point of preventing soil erosion in practical?”. Although it is another story, we changed the duplicate use of “Therefore” to “Finally”.-----Comment 2:Then, in the second paragraph, you can talk about how to do that (no tillage, putting residues, incorporating organic matter etc.). Also at the end, or the next paragraph, you can talk about what is still lacking and needs to be investigated to show the originality of this report.MO: We agree with you. The second paragraph had not followed the previous paragraph. We think the problem is the first sentence and the last sentence. We deleted the first sentence and added the following sentence at the end of the paragraph: “The effect of residue incorporation is unclear”.-----Comment 3:In the last paragraph of the introduction, you put your research aim. 1), and 2) are understandable, but the last sentence is not clear. Why can \"ensuring growth of different crops for the same amount of input residue with different nutrition levels and different soil moisture levels\" provide Response for those two questions?MO: Yes, we agree with you. We need to describe more clearly the aim of the treatment rather than the treatment itself here. We revised the sentence as follows: “we determined water infiltration rates for different biomass levels under even amounts of residue incorporation in crop rotation.”-----Comment 4:Methods:You have N fertilizer treatment, and \"soil moisture\" treatment, but putting as \"three soil moisture levels (un-mulched, ....)\" is not really exact naming for your treatment, since you are not really controlling soil moisture alone (covering with mulch has a lot more effect other than soil moisture). Probably \"mulching treatment\"?MO: You are right. We changed to “mulching treatment”. We are also thinking about the same thing that you have mentioned. We added the following sentence at the end of the first paragraph: \"Although both nitrogen application and mulch treatment have some impacts, we expected the changes in top-root ratios.\".-----Comment 5:Second paragraph: Please put the information in order. You are talking about the end of Okura cropping, then going back to the corn cropping next. Please make it easy for readers to understand by putting them in logical manner.MO: We added a supplemental figure of the timeline. We moved the first sentence to the determination section.-----Comment 6:\"we repeated the above processes\": What are the processes? There are so many processes in \"above\". Do you mean all including corn cropping?MO: Thank you for the suggestion. We cut the word “above” and added the “using rose grass residue” after the word “okra”.-----Comment 7:It is not clear whether you returned residues evenly among plots within the house, or across the houses.MO: See the sentence “(each plot received the same amount of residue for the next crop per house; the amount was different between the houses)\". We think many readers will have the same question; however, now the added sentence at the end of the introduction and the supplemental figure will help their understanding.-----Comment 8:Data analysis: Since this experimental design is 2 factor, 3 levels, randomized block design with 2 replication (block), please conduct 2-way anova accordingly.MO: This study conducted correlation analysis, and the strong correlation means significance. We believe, anova is not needed because the correlation analysis of this study gives much information.-----Comment 9:Results, Discussion and conclusions:If you use the 2-way ANOVA results, you might find something interesting. Since the residue amount increases as the crop biomass and crop root biomass increases, these results are basically showing the same thing. When you had more residue (for example, when you have grown rose grass), you also had more above ground and root biomass. So the Figure 1a result is concomitant with the crop biomass (and root biomass), and not purely showing the effect of the residue amount. If you want to compare the residue incorporation effect and crop biomass (and root biomass), you need to have plots with uniform root biomass condition, and incorporate residues in different levels.MO: Thank you for the important question! You mean that both the amount of residue and the above ground biomass is significant, don't you? We knew. We also knew they are concomitant. However, how do you think about the outlier? To tell the truth, we had expected that returning plant residue is effective for preventing soil erosion; however, it denied in another experiment (unpublished data). “the average infiltration rate of initial stage was almost the same as that of after okra, although the input quantity of the initial stage (2.0 Mg ha−1) was a 2.5-fold higher than after okra (0.8 Mg ha−1)”. The DM amount, 2.0 Mg ha−1 is a considerable amount. The necessity of having plots with “uniform root biomass” is quite agreeable for us too. Therefore, we titled our research as “may affect”. This Research note discusses only unexpected results that come from an experiment that was carried out by our resources."
}
]
}
] | 1
|
https://f1000research.com/articles/7-1523
|
https://f1000research.com/articles/9-164/v1
|
05 Mar 20
|
{
"type": "Systematic Review",
"title": "Comparison of the Informed Health Choices Key Concepts Framework to other frameworks relevant to teaching and learning how to think critically about health claims and choices: a systematic review",
"authors": [
"Andrew D. Oxman",
"Laura Martínez García",
"Laura Martínez García"
],
"abstract": "Background: The Informed Health Choices (IHC) Key Concepts are principles for evaluating the trustworthiness of claims about treatment effects. The Key Concepts provide a framework for developing learning-resources to help people use the concepts when treatment claims are made, and when they make health choices. Objective: To compare the framework provided by the IHC Key Concepts to other frameworks intended to promote critical thinking about treatment (intervention) claims and choices. Methods: We identified relevant frameworks from reviews of frameworks, searching Google Scholar, citation searches, and contact with key informants. We included frameworks intended to provide a structure for teaching or learning to think critically about the basis for claims, evidence used to support claims, or informed choices. For a framework to be included, there had to be a description of its purpose; a list of concepts, competences, or dispositions; and definitions of key terms. We made independent assessments of framework eligibility and extracted data for each included framework using standardised forms. Results: Twenty-two frameworks met our inclusion criteria. The purpose of the IHC Framework is similar to that of two frameworks for critical thinking and somewhat similar to that of a framework for evidence-based practice. Those frameworks have broader scopes than the IHC Framework. An important limitation of broad frameworks is that they do not provide an adequate basis (concepts) for deciding which claims to believe and what to do. There was at most some overlap between the concepts, competences, and dispositions in each of the 22 included frameworks and those in the IHC Framework. Conclusions: The IHC Key Concepts Framework appears to be unique. Our review has shown how it and other frameworks can be improved by taking account of the ways in which other related frameworks have been developed, evaluated, and made useful.",
"keywords": [
"critical thinking",
"evidence-informed decision-making",
"evidence-based practice",
"evidence informed decision-making",
"argumentation",
"causal inference",
"cognitive biases",
"epistemic cognition",
"health literacy",
"logical fallacies",
"meta-cognition",
"scientific thinking",
"frameworks",
"models",
"concepts",
"competences",
"concepts"
],
"content": "Introduction\n\nClaims about what people can do to improve or protect their health (treatments) are ubiquitous. They are found in the mass media, advertisements, and everyday personal communication. Some are based on trustworthy evidence. Many are not, and many people have difficulties determining which claims to believe and act on. Acting on untrustworthy claims and not acting on ones that are trustworthy can result in unnecessary suffering and waste.\n\nIn response to these challenges, we developed the Informed Health Choices (IHC) Key Concepts as the first step in the IHC project1–4. The aim of the IHC project is to help people, particularly primary and secondary school students, learn to assess treatment claims and make informed health choices5.\n\nWe use ‘treatment’ to refer to any intervention or action intended to protect or improve health6. People in other fields have found the IHC Key Concepts relevant for assessing claims about the effects of other types of interventions7. This includes agricultural, educational, environmental, management, social welfare, economic, international development, nutrition, policing, and veterinary interventions.\n\nThe IHC Key Concepts provide a framework for designing curricula, learning resources, and evaluation tools5,8. We first published the framework in 20151 and have continued to update it yearly. The current (2019) framework includes 49 concepts in three groups (Table 1), 20 competences in four groups (Table 2), and 16 dispositions in four groups (Table 3)4. The concepts are principles for evaluating the trustworthiness of treatment claims and the evidence used to support these, and for making informed choices. The methods used to develop the framework are described elsewhere1,3. The framework is a starting point to help teachers, journalists, researchers and other intermediaries to identify and develop resources to help people learn to assess treatment claims and make informed choices.\n\n*A good decision is one that makes effective use of the information available to the decision maker at the time the decision is made. A good outcome is one that the decision maker likes. The aim of thinking critically about treatments is to increase the probability of good outcomes (and true conclusions), but many other factors affect outcomes aside from critical thinking36.\n\nThere are many other frameworks that include concepts, competences, or dispositions that are relevant to thinking critically about treatment claims, comparisons, and choices. These include critical thinking frameworks, logical fallacies and argumentation frameworks, cognitive frameworks, frameworks for scientific thinking, and frameworks related to evidence-based health care. For each category of frameworks there are disagreements about definitions and what is included. For example, learning to think critically is widely held as an aim of education9, but there is not agreement on the definition of “critical thinking” and there are several different frameworks (conceptual structures intended to serve as a support or guide) for critical thinking10–14. Similarly, there are different definitions and frameworks for scientific thinking (reasoning and literacy)15–18, epistemic cognition and meta-cognition19,20, health literacy21–23, and various aspects of evidence-based health care24–26. There is also overlap across these different framework categories, some of which have been grouped together as frameworks for “productive thinking”12.\n\nDefinitions of terms that we use in this paper are shown in Table 4.\n\n\nObjective\n\nThe objective of our review was to systematically compare the IHC Key Concepts Framework to other frameworks that are relevant to teaching and learning how to think critically about treatment claims, evidence, and choices. We examined similarities and differences between the IHC Key Concepts Framework and other frameworks - particularly in the context of primary and secondary school education - including:\n\nThe purposes and definitions of key terms\n\nThe elements included and domains in which they are grouped\n\nHow the frameworks have been developed and evaluated\n\nHow the frameworks have been used to develop curricula, teaching and learning resources, and assessment tools\n\n\nMethods\n\nWe conducted a systematic review of frameworks relevant to teaching and learning to think critically about treatment claims, evidence used to support those claims, and choices. The protocol for the review is published on our website27.\n\nWe included frameworks that are intended to provide a structure for teaching or learning to think critically about at least one of the following:\n\nThe basis (justification) for claims or arguments about the effects of interventions and the reliability of those justifications\n\nThe extent to which evidence used to support claims about the effects of interventions (comparisons) is fair and reliable\n\nChoices about what to do in order to achieve a goal\n\nTo be included, the sources for each framework had to include:\n\na description of the purpose of the framework;\n\na list of the framework’s elements; and\n\ndefinitions of the key terms used to describe the purpose of the framework, its elements and domains (in which elements are grouped, if there are any).\n\nFrameworks that are modifications of another framework were considered together with the framework that had been modified.\n\nWe began by considering 41 frameworks reviewed in Frameworks for Thinking: A Handbook for Teaching and Learning12 and frameworks with which we were already familiar21–35. We searched for other relevant frameworks using Google Scholar between October 2018 and June 2019 using the search strategies found in Extended data File 1. We supplemented these searches by conducting citation searches and contacting key informants for each category of the frameworks.\n\nOne review author (ADO) initially screened frameworks for possible inclusion. Both review authors then independently assessed full-text articles for each potentially relevant framework using an eligibility form (Extended data File 2). We discussed disagreements and reached a consensus. Frameworks that were assessed for inclusion by both authors and then excluded are listed with the reasons for exclusion in Table 5.\n\nFor each included framework, we compiled a list of publications that describe the framework, its development and evaluation, and its use as the basis for curricula, learning resources, and assessment tools.\n\nWe recorded independently the following information for each framework, using a data collection form (Extended data File 3):\n\nIts purpose\n\nIts domains and elements\n\nDefinitions of key terms used to describe its purpose, domains, or elements\n\nMethods used to develop the framework\n\nMethods used to evaluate the framework (if any), and findings\n\nWays in which the framework has been used as the basis for\n\nCurricula\n\nTeaching and learning\n\nAssessment tools\n\nWe compared the data that each of us had collected, discussed disagreements, and reached a consensus.\n\nBased on this information, we assessed independently:\n\nstrengths and weaknesses of how each framework had been developed and evaluated\n\nstrengths and weaknesses of how each framework has been or could be used\n\nany other strengths or weaknesses\n\nWe compared our assessments, discussed disagreements, and reached a consensus.\n\n1. We summarised key characteristics of the included frameworks in tables.\n\n2. Using Venn diagrams, we mapped the extent to which the purposes of the different frameworks overlap with those of the IHC Key Concepts Framework.\n\n3. We compared the concepts, competences and dispositions in each framework with those in the IHC Key Concepts Framework. We considered separately any elements that could not be categorised as concepts, competences or dispositions.\n\n4. We reflected on our assessments of the frameworks and identified implications for how we might improve the IHC Key Concepts Framework, and its usefulness.\n\nWe conducted these analyses independently and then compared our analyses, discussed disagreements, and reached consensus.\n\n\nResults\n\nWe screened over 1600 references retrieved using Google Scholar (search strategy: Extended data File 1). In addition, we screened the reference lists in the articles that we retrieved. We identified over 80 frameworks and assessed 35 of these for eligibility based on one or more full-text articles (Figure 1). We excluded 13 of these (Table 5), so ended up including 22 frameworks (Table 6).\n\nFrameworks that we grouped together (e.g. health literacy frameworks) are counted as single frameworks. † Frameworks for Thinking: A Handbook for Teaching and Learning (Mosely 2005) has 41 frameworks. ‡ Our primary Google Scholar searches yielded 1588 records. § These frameworks were excluded after being scanned by one of the review authors (ADO).\n\nWe included four frameworks on critical thinking, three on logic and argumentation, four on cognition, four on scientific thinking, and seven on evidence-based healthcare. We grouped several frameworks together for five types of frameworks - logical fallacies, cognitive biases, epistemological models, systems thinking, and health literacy. We also considered related frameworks together with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework and the Cochrane Risk of Bias Tool. The purpose and background of each of the included frameworks are shown in Table 6, and definitions of the key term for each framework are shown in Table 7.\n\nWe summarise our comparison of the included frameworks to the IHC Key Concepts Framework in Table 8. Two frameworks had a similar purpose: Ennis’ taxonomy of critical thinking dispositions and abilities12,50–59 and Baron’s model of the good thinker12,36,69–75. Ennis’ goal is for students to learn to think critically about what to believe or do. Baron’s goal is for students to learn to think more rationally, that is, in a way that helps them to achieve their goals. Both those goals are broader than that of the IHC Key Concepts Framework, which is to enable people to make informed decisions about which claims to believe about the effects of things they can do (interventions) for their health, the health of others or for other reasons, and about what to do to achieve their goals4. The purposes of the two other critical thinking frameworks that we included (the Model of critical thinking and List of critical thinking skills) were also somewhat like the purpose of the IHC Key Concepts Framework.\n\n† Yes = included in the framework; No = not included in the framework\n\nFigure 2 illustrates how we view the relationship between critical thinking and the IHC Key Concepts Framework. Although the IHC framework focuses specifically on critical thinking about health effects and choices, the same Key Concepts can be applied to many other types of interventions (actions) and decisions7. Because achieving our goals depends on what we do (actions), deciding what to believe about the possible effects of our actions and what to do is at the centre of critical thinking. However, critical thinking also applies to many other types of beliefs, such as beliefs about religion, history, or art.\n\nThe goal of the IHC Key Concepts Framework is “To enable people to make good decisions about which claims to believe about the effects of things they can do for their health, the health of others or for other reasons, and about what to do to achieve their goals”4. Our formulation of that goal was influenced by how Ennis and Baron formulated their goals. We have adapted Baron’s definition of a “good decision”36 to explain what this means: a good decision is one that makes effective use of the information available to the decision maker at the time the decision is made. A good outcome is one that the decision maker likes. The aim of thinking critically about treatments is to increase the probability of good outcomes (and true conclusions), but many other factors affect outcomes aside from critical thinking.\n\nThe purpose of one of the logic and argumentation frameworks that we included had a somewhat similar purpose to that of the IHC Key Concepts Framework. The evidence-based reasoning framework87 was developed as an analytic tool intended as a foundation for assessing students’ ability to reason from evidence in writing and classroom discussions. The relationship between argumentation – critical evaluation of arguments – and the IHC Key Concepts Framework is illustrated in Figure 3. The purposes of four of the evidence-based health care frameworks were also somewhat similar to the purpose of the IHC Key Concepts Framework: health literacy21–23,145–149, the Evidence-based practice (EBP) core competencies26, GRADE25,30,154–160, and critical appraisal tools29,166–179.\n\nFigure 4 illustrates the relationship between the evidence-based practice framework and the IHC Key Concepts Framework. Evidence-based practice is a framework for health professionals, whereas the IHC Key Concepts Framework is for young people, patients and the public, and policymakers, as well as health professionals. Evidence-based practice is a broader framework, which includes critical appraisal of other types of evidence besides evidence of effects. It also includes formulating clinical questions, acquiring evidence, and evaluating performance, which are largely outside of the scope of the IHC Key Concepts Framework. The aim of evidence-based practice is to improve health outcomes, and that depends on what health professionals, patients and the public do. Thus, the IHC Key Concepts Framework – critical thinking about effects and choices – is at the centre of evidence-based practice, in much the same way as it is at the centre of critical thinking.\n\nHealth literacy also has a broader focus than the IHC Key Concepts Framework. This is most clearly illustrated by Nutbeam’s framework145,148, which divides health literacy into functional, interactive, and critical health literacy. The IHC Key Concepts Framework is most closely related to critical health literacy, as illustrated in Figure 5.\n\nThe GRADE framework overlaps substantially with the IHC Framework with respect to critical thinking about evidence of intervention effects and decisions about what to do, as illustrated in Figure 6. However, the GRADE framework is designed primarily for judgements by authors of systematic reviews, guideline developers, and policymakers.\n\nLogical fallacies35,68,76–84 and cognitive biases36,88–96 are both highly relevant to the IHC Key Concepts Framework. However, there is little similarity between the purposes of either of those types of frameworks and the purpose of the IHC Key Concepts Framework (Figure 7 and Figure 8). Recognising the use of faulty reasoning in the construction of an argument overlaps with recognising faulty logic underlying claims about effects. However, most logical fallacies are not directly relevant to this. Similarly, recognising systematic patterns of deviation from rational judgements (cognitive biases) overlaps with judgements about effects and choices, but most cognitive biases are not directly relevant. In addition, most of the IHC Key Concepts are not logical fallacies or cognitive biases.\n\nThere was at most some overlap between the concepts, competences, and dispositions in the included frameworks and those in the IHC Key Concepts Framework (Table 8). In seven of the 16 frameworks that included concepts, there was some overlap with the IHC Key Concepts Framework. Of the 13 frameworks that included competences, there was some overlap with the IHC Key Concepts Framework in five. There was very little overlap with the dispositions included in eight frameworks.\n\nThe methods used to develop the frameworks were clearly described for only 10 of the 22 included frameworks, and the basis was clear for only six (Table 9). In total, 11 of the 22 were based in part on another framework, three on a model or theory, four on a systematic review, nine on an unsystematic review, three on a formal consensus process, and seven on an informal consensus process. The evidence-based practice core competences and Cochrane Risk of Bias Tool were the most systematically developed frameworks. Both were based in part on systematic and unsystematic reviews. The evidence-based practice core competences used a formal consensus process, whereas the Cochrane Risk of Bias Tool used an informal process.\n\n* More than one framework was considered.\n\n† Although more than one framework was considered, the assessment applies to this specific framework\n\n‡ Yes or yes for some for “clear methods”; yes for other bases\n\nKey findings of formal and informal evaluations of the included frameworks are summarised in Table 10. We found formal evaluations of seven of the 22 included frameworks. Methods used to formally evaluate the frameworks included factor analysis143,146,187; extensive feedback (including online surveys)32; principal components and Rasch analysis143; systematic reviews24,29,157,175,181,185,188; an agreement study189 and an assessment of the effect of training on reliability182; and an assessment of usability using focus groups and online surveys183. Two frameworks were evaluated both formally and informally, were found to be useful, and are widely used: the GRADE framework24,157,188,189 and the Cochrane Risk of Bias Tool181–183,184,185.\n\nOur assessment of the elements (concepts, competences or dispositions) in the 22 frameworks is summarised in Table 11. Only one framework, the framework for K-12 science education, had clear inclusion criteria for one of three dimensions (“core ideas”). We judged the elements to be coherent in five frameworks, distinct in nine, and organised logically in eight. There were no inappropriate elements in seven frameworks and no missing elements in two. Overall, the evidence-based reasoning framework86 was the only framework that we assessed positively for all five criteria (coherent elements, distinct elements, no inappropriate elements, no missing elements, and logical grouping of the elements). That framework is a relatively simple analytic model of arguments about scientific ideas.\n\n* More than one framework was considered.\n\n† Although more than one framework was considered, the assessment applies to this specific framework\n\n‡ Yes or yes for some for “clear methods”; yes for “coherence”, “distinct”, and “logical grouping”; no for “inappropriate elements” and “missing elements”\n\n§ Does not mix type(s) and specificity of concepts, competencies, or dispositions\n\n** Included concepts, competencies, or dispositions are clearly different from each other\n\n†† Concepts, competencies, or dispositions included in the framework that should not have been\n\n‡‡ Concepts, competencies, or dispositions not included in the framework that should have been\n\n§§ Concepts, competencies, or dispositions organised in a way that makes sense\n\nInformation about how the 22 frameworks have been used is summarised in Extended data File 4. We found evidence that most of the frameworks were being used. For four (the taxonomy of concepts and critical abilities related to the evaluation of verbal arguments, the evidence-based reasoning framework, the AIR model of epistemic cognition, and the model for scientific thinking) we found little evidence of use. Two had only been available for one or two years (the evidence-based practice core competences and the Catalogue of Biases), and we were uncertain about their use. Twelve of the frameworks appeared to be intended primarily for teachers and students, and we found learning resources based on 14 of the frameworks.\n\nNine of the frameworks appeared to be intended primarily for researchers. One (the evidence-based practice core competences) appeared to be intended primarily for curriculum developers26. We found at least some evidence that six other frameworks were used for curriculum development, including three of the critical thinking frameworks. We found evidence that 12 of the frameworks were used as the basis for one or more assessment tools. Other ways in which the frameworks have been used or have been proposed for use include: self-teaching; by parents, institutions, and government; by employers developing training programs; professional development; establishing norms or standards; developing ways of protecting against cognitive biases; theory development; intervention design; policy advice; and reporting standards.\n\nStrengths and weaknesses of each framework and ideas for further development of the IHC Key Concepts Framework are summarised in Table 12. Strengths of the frameworks related to their development include international collaboration, support from international or national organisations, continued development over a long period of time, well described and systematic development, research evidence to support all of the concepts, elicitation of extensive feedback, and formal comparisons to similar frameworks. Strengths related to their usability include simplicity, a user-friendly structure for describing each concept, and wide use.\n\nWeaknesses of the frameworks include unclear development methods, lack of formal evaluation, multiple frameworks with the same focus and no apparent agreement or effort to reach a consensus on an optimal framework, and complexity or many included concepts or competences.\n\nWe identified several ways in which the IHC Key Concepts Framework might potentially be improved (Table 12). These include making the evidence that supports each IHC Key Concept explicit, including evidence of the extent to which each IHC Key Concept is not widely understood or applied; designing a website to popularise teaching and learning about, understanding of, and application of the IHC Key Concepts Framework; and developing a visual model of the IHC Key Concepts Framework.\n\nOverall, our review of the concepts, competences, and dispositions in the 22 frameworks led us to add four new concepts to the IHC Key Concepts Framework, to modify 16, and to add 10 new competences and four new dispositions4.\n\n\nDiscussion\n\nWe identified 22 frameworks that overlap with the IHC Key Concepts Framework. We found that the purpose of the IHC Key Concepts Framework is most like two frameworks for critical thinking: Ennis’ taxonomy of critical thinking dispositions and abilities and Baron’s model of the good thinker. However, in terms of concepts and competences, there was more overlap with Halpern’s list of critical thinking skills. Although the IHC framework drew on evidence-based health care frameworks, there was at most some similarity with the purposes of those frameworks and the purpose of the IHC Key Concepts Framework. There was some overlap in terms of concepts with GRADE, critical appraisal tools, and the Catalogue of Bias. There was overlap in terms of competences with health literacy, the evidence-based practice core competences, and critical appraisal tools.\n\nWe found the IHC Key Concepts Framework to be central to critical thinking and evidence-based practice, both of which have broader scopes than the IHC Key Concepts Framework. An important weakness we found with these and other broad frameworks, such as those that focus on argumentation, is that they do not provide an adequate basis (concepts) for thinking critically about claims about the effects of interventions and decisions about what to do. As noted by Dewey: “It would be impossible to over-estimate the educational importance of arriving at conceptions: that is, meanings that are general because applicable in a great variety of different instances in spite of their difference. They are known points of reference by which we get our bearings when we are plunged into the strange and unknown. Without this conceptualizing, nothing is gained that can be carried over to the better understanding of new experiences”196. The IHC Key Concepts are applicable to a great variety of claims about the effects of interventions, not just health interventions7, and they are essential points of reference for deciding which claims to believe and what to do.\n\nWe did not find any overlap between the IHC Key Concepts and those included in the framework for K-12 science education, and little overlap in the competences. That framework places little focus on applied science, practical understanding and use of science by non-scientists, and what children will remember and make use of in their daily lives. This may be the case for many national science curricula.\n\nOur review has helped us to clarify the goal of the IHC Key Concepts Framework and led us to add four new concepts, 10 new competences, and four new dispositions. In addition, we have identified ways in which we can improve the methods we use to further develop and evaluate the IHC Key Concepts Framework and make it more useful.\n\nPrevious systematic and unsystematic reviews have reviewed different types of frameworks with similar purposes, including frameworks for cognitive biases190, epistemic cognition42, health literacy22, assessments of the certainty of evidence and recommendations or decisions24,157,193, causal inference162, critical appraisal29, and assessment of the risk of bias181,185. Moseley and colleagues12 conducted a comprehensive review of frameworks for thinking, which overlaps with and informed our review. However, we are unaware of other reviews with the same scope as this review, whether in terms of the included frameworks or the data that were collected for each included framework.\n\nWe used explicit inclusion criteria for frameworks and two review authors independently collected data from included frameworks using a data collection form. Both the eligibility assessments and the data collection required judgement. Although we frequently disagreed, most of our disagreements were minor and all our disagreements were easily resolved. We did not conduct an exhaustive search for relevant frameworks. There may be other frameworks that meet our inclusion criteria. It is possible that other frameworks could add to our findings, but unlikely that they would otherwise substantially change the findings of this review.\n\n\nConclusions\n\nAs defined by Moseley and colleagues: “Framework is a general term for a structure that provides support”12. We have systematically considered 22 frameworks that are relevant to supporting critical thinking about claims about the effects of interventions (actions), comparisons (evidence used to support those claims), and decisions about what to do. We have found that the IHC Key Concepts Framework is unique and that it can be improved by building on the ways in which other related frameworks have been developed, evaluated, and made useful. Much of what we have found can also inform the development and evaluation of other frameworks.\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\nNorwegian Centre for Research Data, Enabling Sustainable Public Engagement in Improving Health and Health Equity, https://doi.org/10.18712/NSD-NSD2817-V1197.\n\nThis project contains the following extended data:\n\n- File 1: Search strategy\n\n- File 2: Critical thinking frameworks eligibility form\n\n- File 3: Critical thinking frameworks data collection form\n\n- File 4: Use of the frameworks\n\n- File 5: PRISMA checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe PRISMA checklist for ‘Comparison of the Informed Health Choices Key Concepts Framework to other frameworks relevant to teaching and learning how to think critically about health claims and choices: a systematic review’, https://doi.org/10.18712/NSD-NSD2817-V1197.\n\nInformation about this dataset can be found in English here: http://nsddata.nsd.uib.no/webview/index.jsp?v=2&submode=ddi&study=http%3A%2F%2Fnsddata.nsd.uib.no%2Fobj%2FfStudy%2FNSD2817&mode=documentation",
"appendix": "Acknowledgments\n\nWe would like to thank Astrid Dahlgren, Iain Chalmers, and Joe Chislett for comments on the protocol; Steve Higgins for advice; and Iain Chalmers, Matt Oxman, and Sarah Rosenbaum for comments on a draft of this report.\n\n\nReferences\n\nAustvoll-Dahlgren A, Oxman AD, Chalmers I, et al.: Key concepts that people need to understand to assess claims about treatment effects. J Evid Based Med. 2015; 8(3): 112–25. PubMed Abstract | Publisher Full Text\n\nChalmers I, Oxman AD, Austvoll-Dahlgren A, et al.: Key Concepts for Informed Health Choices: a framework for helping people learn how to assess treatment claims and make informed choices. BMJ Evid Based Med. 2018; 23(1): 29–33. PubMed Abstract | Publisher Full Text\n\nOxman AD, Chalmers I, Austvoll-Dahlgren A, et al.: Key Concepts for assessing claims about treatment effects and making well-informed treatment choices [version 2; peer review: 3 approved]. F1000Res. 2019; 7: 1784. 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Int J Epidemiol. 2002; 31(2): 422–9. PubMed Abstract | Publisher Full Text\n\nPhillips CV, Goodman KJ: The missed lessons of Sir Austin Bradford Hill. Epidemiol Perspect Innov. 2004; 1(1): 3. PubMed Abstract | Free Full Text\n\nSchünemann H, Hill S, Guyatt G, et al.: The GRADE approach and Bradford Hill's criteria for causation. J Epidemiol Community Health. 2011; 65(5): 392–5. PubMed Abstract | Publisher Full Text\n\nCritical Appraisals Skills Programme: CASP Checklist: 11 questions to help you make sense of a Case Control Study. Accessed November 27, 2019. Reference Source\n\nCritical Appraisals Skills Programme: CASP Checklist: 12 questions to help you make sense of a Cohort Study. Accessed November 27, 2019. Reference Source\n\nCritical Appraisals Skills Programme: CASP Checklist: 11 questions to help you make sense of a Randomised Controlled Trial. Accessed November 27, 2019. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nCharnock D, Shepperd S: Learning to DISCERN online: applying an appraisal tool to health websites in a workshop setting. Health Educ Res. 2004; 19(4): 440–6. PubMed Abstract | Publisher Full Text\n\nHyde C, Parkes J, Deeks J, et al.: Systematic review of effectiveness of teaching critical appraisal. Oxford, UK: Institute of Health Sciences, Centre for Statistics in Medicine, 2000. Reference Source\n\nOxman AD, Sackett DL, Guyatt GH: Users' guides to the medical literature. I. How to get started. The Evidence-Based Medicine Working Group. JAMA. 1993; 270(17): 2093–5. PubMed Abstract | Publisher Full Text\n\nShaneyfelt T, Baum KD, Bell D, et al.: Instruments for evaluating education in evidence-based practice: a systematic review. JAMA. 2006; 296(9): 1116–27. PubMed Abstract | Publisher Full Text\n\nAmerican Medical Association, Centre for Health Evidence: Summarizing the Evidence. Learning Tools. Accessed November 27, 2019. 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}
|
[
{
"id": "73960",
"date": "03 Nov 2020",
"name": "Declan Devane",
"expertise": [
"Reviewer Expertise Evidence syntheses",
"randomised trials",
"maternity care"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this paper have done a good job in conducting a systematic review of frameworks that overlap with, or potentially overlap with, the IHC key concepts framework.\nThe Informed Health Choices (IHC) Key Concepts Framework may help build an early foundation for thinking critically about health claims and learning to make decisions informed by evidence. It provides a potential means of going beyond the delivery of information, to the development of critical thinking skills. There are however other frameworks that are relevant to thinking critically about treatment claims, comparisons, and choices. In this paper the authors position the theoretical underpinnings of the IHC Key Concepts Framework in existing frameworks within the fields of health literacy and critical thinking.\nSystematic review methods are detailed and appropriate as are process for decision making. The authors did not conduct an exhaustive search. It is possible therefore that additional frameworks may have been excluded. the authors acknowledge this limitation and I agree with them in that it is unlikely that additional frameworks would substantially change the findings of their review.\nComparisons between the IHC framework and the included frameworks in the review are tabulated clearly and the accompanying figures demonstrating relationships between, for example, critical thinking and the key concepts framework are welcomed. It is refreshing to see that the findings of the review led the authors to revise their framework by adding concepts, competencies and dispositions.\nAlthough this paper is focused largely on the context of primary and secondary school education, it has learning for use of the IHC framework outside of these contexts and also for the development and evaluation of other frameworks. The paper also offers a useful structure for cross comparisons of frameworks in any setting or context.\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": "73961",
"date": "16 Nov 2020",
"name": "Loai Albarqouni",
"expertise": [
"Reviewer Expertise Evidence-based Healthcare"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 inviting me to review this interesting systematic review. The authors of this article systematically review and compare frameworks relevant to teaching and learning of critical thinking about claims, evidence, and choices. This is a great article that is tackling a very interesting issue related to the concepts of critical thinking. The authors elegantly visualised the overlap between IHC frameworks and other frameworks on a group of Venn diagrams and included a list of definitions and terminologies that are very useful. The authors provide a detailed transparent description of their methods and results – very impressive. A couple of comments:\nAuthors might consider describing how they collected the data regarding the use of these frameworks as the basis for curricula/teaching & learning/ assessment tools – as this usually goes beyond the framework publications.\n\nAuthors mentioned that as a result of this work, they made a few modifications/additions to the original IHC concepts – authors might consider describing/justifying these changes.\nI would like to congratulate the authors on this great work.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "74232",
"date": "30 Nov 2020",
"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\nThanks to the authors for this important and interesting paper.\nThe Informed Health Choices (IHC) project aims to support people to evaluate the trustworthiness of health claims and to make informed choices. The IHC Key Concepts provide a framework for designing curricula, learning resources and evaluation tools. The framework has been updated and extended regularly. It comprises several concepts, competences and dispositions in different groups.\nObjective of this systematic review was to compare the IHC Key Concepts Framework to other frameworks that are relevant to teaching and learning how to think critically about treatment claims, evidence and choices.\nThe frameworks were mainly identified from reviews of frameworks and by searching Google Scholar. Twenty-two frameworks were included. Two authors independently extracted information on purposes, definitions of key terms, included elements, methods of development and evaluation, and the way the frameworks were used to develop curricula, learning resources and assessment tools. Strengths and weaknesses of each framework were assessed. The authors described the frameworks in detail and displayed differences and similarities in comparison to the IHC Key Concepts Framework. They concluded that the IHC Key Concepts Framework is unique and that it can be improved by taking account of the ways in which other related frameworks have been developed, evaluated, and made useful. The findings can also be used to improve other frameworks.\nA possible limitation of the manuscript is the exclusively search in Google Scholar. Underlying algorithms are unknown and searches cannot be replicated reliably. The authors themselves stated that they may have missed frameworks. We agree with them that it is unlikely that additional frameworks would substantially change the findings of the review.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-164
|
https://f1000research.com/articles/9-163/v1
|
04 Mar 20
|
{
"type": "Research Article",
"title": "Surveillance of neonatal herpes in the British Isles 2004-2006",
"authors": [
"Pat A Tookey",
"Saboura Mahdavi",
"Catherine S Peckham",
"Saboura Mahdavi",
"Catherine S Peckham"
],
"abstract": "Background: Neonatal herpes simplex virus (HSV) infection is rare but potentially devastating and can result in neonatal death or serious disability. National incidence was estimated at 1.65/100,000 live births in an earlier British Paediatric Surveillance Unit (BPSU) study of births 1986-1991. Methods: A second surveillance study of neonatal HSV was undertaken through the BPSU 2004-2006, with follow-up information collected on surviving children in early childhood. Results: Over the three-year period, 85 infants were reported with confirmed neonatal HSV, an estimated incidence of 3.58/100,000 live births (95% CI 2.86-4.42), about double that reported almost two decades earlier. Over 40% of infants were pre-term compared with 25% in the earlier period. Just over 70% had central nervous system (CNS) or disseminated infection, and among these 54% had no skin, eye or mouth lesions noted. Almost all received antivirals, but 22 (26%) neonates died, all with disseminated or CNS infection. All but six infections were typed, of which 57% involved HSV-2; the increased risk of adverse outcomes associated with HSV-2 in the earlier study was confirmed and strengthened, with twice as many deaths or long term disability in infants with HSV-2 than HSV-1. As before, a reported history or diagnosis of maternal HSV infection was rare prior to infant diagnosis. Likely timing of infant exposure to HSV could only be assigned in 43% of cases, of which just over half were probable postnatal transmissions. Conclusions: Neonatal HSV infection remains rare although incidence doubled in the British Isles between the late 1990s and the mid-2000s. These findings suggest that future research should explore the relationship between pre-term delivery and infant susceptibility, and also the role of postnatal acquisition of infection. Healthcare professionals and new parents must continue to be aware of this rare condition in order to enable prompt investigation and instigation of treatment.",
"keywords": [
"Neonatal Herpes",
"HSV",
"surveillance",
"neonates"
],
"content": "Introduction\n\nNeonatal herpes simplex virus (HSV) infection is a rare but potentially devastating condition which can follow primary or recurrent maternal infection in pregnancy or be acquired postnatally through direct contact with infected secretions. Transplacental transmission is unusual, and perinatal infection is usually acquired through an infected birth canal1,2.\n\nThe incidence of neonatal HSV infection ranges from about 10 per 100,000 live births in the USA3 to 2–6 per 100,000 in the UK4, Australia5 and Canada6. Maternal infection close to term increases the risk of neonatal infection, and infants whose mothers have primary infection close to delivery are more likely to acquire infection than those whose mothers have recurrent infection7.\n\nAlthough oral infection is predominantly associated with HSV-1, and genital infection with HSV-2, there is considerable crossover8. Reactivation appears to be more frequent following HSV-2 than HSV-1. Prior HSV-1 infection is partially protective against the acquisition of HSV-2 and may prevent the severe clinical manifestations associated with primary infection. Many women who have had genital HSV would not be aware of it, as both primary infection and reactivation can be asymptomatic9.\n\nSurveillance of neonatal HSV was first undertaken through the British Paediatric Surveillance Unit (BPSU) in 1986–1991; estimated incidence of infection was then 1.65/100,000. Neonatal infection was attributed to HSV-1 and HSV-2 in equal proportion, but the virus could not be typed in one third of cases; more than half of the 76 infants reported died, or had substantial disability at follow up4.\n\nIn the light of the increasing prevalence of sexually transmitted diseases and demographic and social changes within the British population, a second BPSU study was carried out 2004–2006, with preliminary results presented in BPSU Annual Reports10 but not elsewhere. To enable comparison over time, and with a third study being instigated through the BPSU, we want to make the final data from the second study more widely available, and highlight the changes occurring in the 20 years between the two completed national studies.\n\n\nMethods\n\nEthics approval for this study was granted by London MREC (reference MREC/03/2/80; PIAG/BPSU 2-10(g)/2005). Cases were notified through the normal BPSU protocols to this study between 2004 and 2006; paediatricians were not required to request patient consent for reporting, cases were pseudonomysed, and the study team had no contact with reported cases.\n\nThis study was carried out through the BPSU’s active national surveillance scheme, established in 1986, whereby consultant paediatricians (mainly members of the Royal College of Paediatrics and Child Health) report specified rare paediatric conditions on a monthly basis. On receipt of a report the BPSU notifies the appropriate study investigator who contacts the reporting clinician for further details. Respondents make a nil return if they have no cases to report; the BPSU monitors response rates, which at the time of this study were 94%. Full details of the BPSU methodology have been described elsewhere11. For this study, paediatricians were asked to report any infant under one month of age with a laboratory-confirmed diagnosis of HSV infection, or any such infant treated with antiviral drugs for suspected HSV infection, or any stillborn infant in whom HSV was suspected, born between 1 January 2004 and 31 December 2006. Respondents were then asked to complete a standard questionnaire providing demographic, clinical and laboratory details including maternal and perinatal information, presentation and treatment. Subsequently, the notifying paediatrician was asked to provide brief follow-up information on the health outcome of the surviving children, including any further treatment required, in their second or third year of life. All clinical and laboratory investigations were undertaken locally as part of the normal care of the infant.\n\nInfants with neonatal HSV confirmed by virus culture, polymerase chain reaction (PCR) or immunofluorescence (IF) on a sample taken within 28 days of birth, were classified as confirmed cases. HSV type was reported by the notifying paediatrician on the basis of laboratory results.\n\nPaediatricians were asked to classify infant presentation as disease localised to the skin, eye and/or mouth (SEM), disseminated infection, with or without central nervous system (CNS) and/or SEM involvement, or CNS infection with or without SEM involvement, as described by Whitley1, and in accord with other similar studies4–6.\n\nAdverse health outcomes among survivors were classified as mild, moderate or severe, based on the latest clinical findings reported to the study.\n\nData were managed in a study-specific Microsoft Access 2002 database (Microsoft Corp., Redmond, Washington, USA) and were analysed using Access and Excel.\n\n\nResults\n\nOverall, 194 reports of neonatal herpes were received, and 85 infants were classified as confirmed cases of neonatal HSV.\n\nOf these, 99 reports were excluded: 53 were duplicates and 46 did not meet the case definition; the latter category included 25 infants treated with acyclovir and reported as suspected cases, but subsequently determined, on the basis of laboratory findings, not to have neonatal HSV. The remaining 10 reports could not be classified as confirmed or excluded as no clinical or laboratory information was provided after the initial case report. This paper focuses on the 85 confirmed cases of neonatal HSV.\n\nA total of 45 boys and 40 girls, including two twin pairs, were reported with confirmed neonatal HSV over the three years. Seventy-one infants were born in England, nine in Scotland, and the remaining five in Wales, Northern Ireland or Ireland, giving a minimum estimated incidence of 3.58/100,000 live births (95% CI 2.86-4.42) in the British Isles. In almost all cases (79, 93%), the virus was typed: 34 (43%) HSV-1, and 42 (53%) HSV-2, with another three infants (4%) reported with dual infection (grouped with those with HSV-2 in this report).\n\nTable 1 shows maternal and infant characteristics. Median maternal age at delivery was 25 years (range 15–45), and only 5% of mothers were reported to be of black or minority ethnic origin. Two sets of twins were delivered vaginally, at 32 and 34 weeks gestation. Just over a third of singletons (35%) were delivered by caesarean section, but in no case was this because of concerns about herpes infection in pregnancy. Median gestational age was 38.5 weeks (range 26–42); 42% of singletons were pre-term (<37 weeks). Median singleton birthweight was 2755 grams (range 948–4700) and all term infants weighed at least 2500 grams at delivery, apart from one born at 37 weeks gestation.\n\nAs shown in Table 2, 61% (52/85) of infants presented with SEM lesions, and for 24, this was the sole manifestation. Of the 61 infants with disseminated or CNS infection, 54% (33) had no SEM lesions reported. Infants with HSV-1 were more likely to have SEM lesions than those with HSV-2 infection (25/34, 74%; 23/45, 51%); CNS and disseminated infection were more common in infants with HSV-2 infection (36/45, 80%; 19/34, 56%).\n\nHSV, herpes simplex virus; NK, not known; SEM, skin, eye and/or mouth; CNS, central nervous system.\n\nMost infants (82/85, 96%) were treated with acyclovir. Two of the three untreated infants died in the second week of life, and were only diagnosed at post-mortem with disseminated infection; the third, with localised infection only, was reported well at age 30 months. Two infants started treatment prior to presentation with clinical symptoms because their mothers were known to have had genital herpes infection in pregnancy (one diagnosed before delivery, one just after); another 23 were treated on the day of presentation with symptoms. The remaining 57 infants started treatment a median two days after presenting with clinical symptoms, 95% within six days. Two infants started treatment more than two weeks after the onset of reported clinical symptoms despite having SEM involvement, but neither had any problems reported at the last follow up. Among the 63 infants who survived the neonatal period, median duration of the initial course of treatment was 14 days, and 45% received subsequent courses of treatment.\n\nIt is known that 23 children (27%) have died, all but one in the neonatal period (Table 3 and Figure 1); all of those who died had disseminated or CNS infection, only five also had SEM lesions, and 45% were pre-term. Of neonates with HSV-2, 15 (33%) of 45 died and six (18%) of 34 with HSV-1 (HSV type unknown for two). Two previously asymptomatic infants who died unexpectedly in their second week of life were diagnosed with disseminated HSV-1 infection at post-mortem; the other 20 neonates who died started treatment a median of one day after presentation with symptoms, 95% within five days; all but one died within 15 days of birth.\n\n* Includes one postneonatal death.\n\n^ Cell includes infant with developmental delay (see Figure 1).\n\nHSV, herpes simplex virus; NK, not known; CNS, central nervous system.\n\nHSV, herpes simplex virus; ms, months.\n\nOf the 63 survivors, 13 (21%) were lost to follow up before their first birthday at a median age of five months: two showed signs of developmental delay when last seen at the ages of one and four months; the remaining 11 had no problems reported at last follow up (Figure 1).\n\nFollow-up information was provided for the remaining 50 children at a median age of 34 months (range 12–65 months). Long-term sequelae were reported in 2/4 surviving children whose virus was not typed, 4/21 (19%) children with HSV-1 infection, and 13/25 (52%) of those with HSV-2. Severe developmental delay and major health problems including recurrent seizures, visual impairment, and quadriplegia or hemiplegia (median age at follow up 37 months, range 24–65) were reported in 13 children, including one child who died aged three years. Another six children had less severe problems reported, including speech and language delay, mild developmental delay and/or visual or hearing impairment (at median age 33 months, range 28–57). The remaining 31 children were reported well or with only minor health issues (at median 33 months, range 12–62).\n\nFor 48 infants (including a pair of twins), it was not possible to determine timing of exposure to HSV, that is, whether they had been exposed prenatally or around the time of delivery to maternal genital infection, or postnatally to an oral cold sore or whitlow. For the other 37 infants, 18 (including a pair of twins) were probably exposed to pre/perinatal maternal genital infection, and 19 to a probable postnatal source (Table 1).\n\nAmong the infants with probable pre/perinatal exposure, the risk was only identified prior to delivery in two cases: one woman, diagnosed with genital HSV during pregnancy, was treated with acyclovir for seven weeks between diagnosis and delivery by emergency caesarean section when she went into early pre-term labour. Another pregnant woman had reported a past history of genital HSV; following a pre-term vaginal delivery her baby was diagnosed with neonatal HSV and subsequently the mother was found to be shedding virus in the genital tract. Two more mothers reported a history of genital herpes prior to pregnancy after their babies were diagnosed. Another thirteen women were retrospectively found to have had symptoms compatible with current or recent genital HSV, including two with partners who were also diagnosed with genital infection.\n\nIn 19 cases, a parent or relative was reported to have a ‘cold sore’ or similar lesion around the time of delivery or infant diagnosis, or to have frequent oral cold sores; this comprised six mothers or fathers with non-genital HSV lesions, five parents who reported that they frequently had oral cold sores, and eight symptomatic visiting relatives.\n\nAmong the 35 infants with type of HSV known and a likely source of infection identified, it was more likely to be postnatal exposure (11/18, 61%) for infants with HSV-1 than for infants with HSV-2 (7/17, 41%). Median gestation was 39 weeks (range 27–42) for infants probably exposed to postnatal infection; 38 weeks (26–42) for infants whose likely exposure was unknown, and 34.5 weeks (27–41) for those with likely pre/perinatal exposure.\n\n\nDiscussion\n\nAlthough reported incidence of neonatal HSV was double that reported in the first BPSU study (3.58 v 1.65 per 100,000, 95% CI 2.9-4.4 v 1.3-2.0) carried out almost two decades earlier4, it remained at a very low level and similar to estimates from the Australian and Canadian Paediatric Surveillance Units5,6. Improvements in diagnostic techniques and clinical awareness may have had an impact on the reported incidence. Although the BPSU is an active reporting system with a high response rate, our estimate can only be a minimum: it is likely that there is an element of under-reporting to this national scheme; in addition, in the later study there were 10 reports of infants with insufficient information to confirm or exclude the diagnosis. However, since the methodology in the first and second BPSU studies was the same, changes over time are likely to be real. Our estimate is also considerably lower than that reported by Batra et al. for the period 2006–2012, based on an urban single centre population in England (17.5 per 100,000, 95% CI 8.4-32.1)12. This could reflect differences in study methodologies, the population under review, or a further increase over time.\n\nIt was encouraging that most infections could be characterised as HSV-1 or HSV-2, in contrast to the earlier BPSU study when around a third could not be typed. Most infants were diagnosed in time to receive antiviral treatment (only three were not treated at all) unlike the earlier study where 20% of infants received no antivirals. There was also only one postneonatal death reported, compared with seven (9%) previously with a similar length of follow up. The observation from the earlier study that a higher risk of adverse outcome is associated with HSV-2 was confirmed and strengthened, with death or long-term morbidity almost twice as common in those with HSV-2 infection as in those with HSV-1. Nonetheless, the overall proportion of adverse outcomes was lower than in the earlier study (overall 51% compared with 58%).\n\nA major difference between this study and the earlier one was the increase in the proportion of pre-term births – over 40% compared with 25%. In this respect our findings concur with those of Batra et al.12. It is possible that pre-term infants are at increased risk of acquiring HSV following perinatal exposure to primary or recurrent maternal infection, due to lack of adequate transfer of maternal antibodies at this stage of gestation13–15. This is an issue which requires further exploration.\n\nAlthough it was not possible to assign a likely source of infection in about 60% of cases, there were notable differences between the infants with probable postnatally acquired infection, who tended to be of normal gestation and more likely to have HSV-1 and SEM involvement, and those with probable pre/perinatal exposure, who tended to be born earlier and to have HSV-2. As with the previous study, a history of past infection or diagnosis prior to delivery was extremely rare, and maternal infection was generally only identified after diagnosis in the infant.\n\nFuture investigations of the national picture with respect to this rare but devastating condition should consider the role of postnatal acquisition and virus type, and the relationship between pre-term delivery and neonatal HSV following both primary and recurrent maternal genital infection. The introduction of screening for HSV susceptibility in pregnancy would not address the issue of infection in pre-term babies exposed to recurrent infection, in view of the lack of transfer of maternal antibody. The important contribution of postnatal acquisition must not be ignored or minimised in the debate around pregnancy screening. The assumption that neonatal herpes is mainly due to maternal genital infection must be challenged – in this study almost a quarter of all cases, and about half of those where a likely source was identified, were attributable to postnatal exposure to cold sores and other herpetic lesions.\n\nThese findings underline the importance of continuing awareness of this rare condition, both among healthcare staff and new parents, with prompt investigation and instigation of treatment.\n\n\nData availability\n\nThe data for this study was collected 2004–2006. At that time, approval to share raw individual patient data was not required or requested. Identifiers including maternal and infant dates of birth and other demographic characteristics were collected as essential study data and adequate anonymisation of data is not possible due to the rarity of the disease. Assurances were provided that all data would be kept securely and not shared outside the study team. Under these circumstances, data cannot be made publicly available, even in an anonymised form. If researchers wish to access these data for the purposes of further research, they may contact the corresponding study author, Dr Pat Tookey, by email (p.tookey@ucl.ac.uk), providing details of the information required and the intended use of the data, for example comparison with other similar case series, and we will do our best to facilitate this.",
"appendix": "Acknowledgements\n\nWe thank the British Paediatric Surveillance Unit (BPSU), supported by the Department of Health, for facilitating the data collection, and acknowledge the invaluable contribution of the reporting clinicians. Dr David Brown (Health Protection Agency) and Richard Lynn (British Paediatric Surveillance Unit) contributed to the design of the study, and Janet Masters (d.2012) worked on data collection and data management. Any views expressed in this paper are those of the authors.\n\n\nReferences\n\nWhitley R: Neonatal herpes simplex virus infections. J Med Virol. 1993; 41(Suppl 1): 13–21. PubMed Abstract | Publisher Full Text\n\nBrown Z: Preventing herpes simplex virus transmission to the neonate. Herpes. 2004; 11(Suppl 3): 175A–86A. PubMed Abstract\n\nFlagg EW, Weinstock H: Incidence of neonatal herpes simplex virus infections in the United States, 2006. Pediatrics. 2011; 127(1): e1–8. PubMed Abstract | Publisher Full Text\n\nTookey P, Peckham CS: Neonatal herpes simplex virus infection in the British Isles. Paediatr Perinat Epidemiol. 1996; 10(4): 432–42. PubMed Abstract | Publisher Full Text\n\nJones CA, Raynes-Greenow C, Isaacs D, et al.: Population-based surveillance of neonatal herpes simplex virus infection in Australia, 1997-2011. Clin Infect Dis. 2014; 59(4): 525–31. PubMed Abstract | Publisher Full Text\n\nKropp RY, Wong T, Cormier L, et al.: Neonatal herpes simplex virus infections in Canada: results of a 3-year national prospective study. Pediatrics. 2006; 117(6): 1955–62. PubMed Abstract | Publisher Full Text\n\nBrown ZA, Wald A, Morrow RA, et al.: Effect of serologic status and cesarean delivery on transmission rates of herpes simplex virus from mother to infant. JAMA. 2003; 289(2): 203–09. PubMed Abstract | Publisher Full Text\n\nScoular A, Norrie J, Gillespie G, et al.: Longitudinal study of genital infection by herpes simplex virus type 1 in Western Scotland over 15 years. BMJ. 2002; 324(7350): 1366–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrugha R, Keersmaekers K, Renton A, et al.: Genital herpes infection: a review. Int J Epidemiol. 1997; 26(4): 698–709. PubMed Abstract | Publisher Full Text\n\nBritish Paediatric Surveillance Unit. 21st Annual Report 2006-2007. 2007.\n\nLynn R, Pebody R, Knowles R: Twenty years of active paediatric surveillance in the the UK and Republic of Ireland. Euro Surveill. 2006; 11(7): E060720.4. PubMed Abstract | Publisher Full Text\n\nBatra D, Davies P, Manktelow BN, et al.: The incidence and presentation of neonatal herpes in a single UK tertiary centre, 2006-2013. Arch Dis Child. 2014; 99(10): 916–21. PubMed Abstract | Publisher Full Text\n\nProber CG, Sollender WM, Yasukawa LL, et al.: Low risk of herpes simplex virus infections in neonates exposed to the virus at the time of vaginal delivery to mothers with recurrent genital herpes simplex virus infections. N Engl J Med. 1987; 316(5): 240–44. PubMed Abstract | Publisher Full Text\n\nPalmeira P, Quinello C, Silveira-Lessa AL, et al.: IgG placental transfer in healthy and pathological pregnancies. Clin Dev Immunol. 2012; 2012: 985646. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimister N: Placental transport of immunoglobulin G. Vaccine. 2003; 21(24): 3365–69. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "90155",
"date": "29 Jul 2021",
"name": "Joan L Robinson",
"expertise": [
"Reviewer Expertise Pediatric 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\nThe authors prevent results from a 2004-2006 active surveillance study for neonatal HSV infection.\nMajor points:\nThe abstract claims that there is increased severity with HSV-2 infection but does not provide data from the study to verify that.\n\nIntroduction – “Although oral infection is predominantly associated with HSV-1, and genital infection with HSV-2, there is considerable crossover.” The reference provided verifies that HSV-1 commonly causes genital disease but I am not aware that there is evidence that HSV-2 causes oral disease. Please provide a reference if this is the case.\n\nMethods – It needs to be clearer which cases were included. The authors state: “For this study, paediatricians were asked to report any infant under one month of age with a laboratory-confirmed diagnosis of HSV infection, or any such infant treated with antiviral drugs for suspected HSV infection, or any stillborn infant in whom HSV was suspected, born between 1 January 2004 and 31 December 2006.” However, later it sounds like all but confirmed cases were excluded. It is not clear why pediatricians were asked to report suspected cases or stillborns with possible HSV if they were going to be excluded unless they had lab confirmation.\n\nResults: “Infants with HSV-1 were more likely to have SEM lesions than those with HSV-2 infection (25/34, 74%; 23/45, 51%); CNS and disseminated infection were more common in infants with HSV-2 infection (36/45, 80%; 19/34, 56%).” Are these differences statistically significant? If so, evidence should be provided.\n\nResults: “Two infants started treatment more than two weeks after the onset of reported clinical symptoms despite having SEM involvement, but neither had any problems reported at the last follow up.” The latter part belongs in the Outcomes section.\n\nResults: “Among the 63 infants who survived the neonatal period, median duration of the initial course of treatment was 14 days, and 45% received subsequent courses of treatment.” What was the range and IQR for duration of treatment? I was surprised that almost half were treated again. Can the authors provide more details? Were any started on acyclovir prophylaxis or was it always treatment?\n\nThe incidence of postnatal exposure is unexpectedly high. The Methods section should explain how this was determined. I disagree with the method that is outlined in the Results section. Most women with genital HSV do not have a history of genital ulcers. Most adults with cold sores do not transmit HSV-1 to a neonate. All prior studies suggested that most cases were transmitted at the time of delivery even if mother had no history of genital lesions. The fact that a visiting relative had a cold sore does not convince me that transmission was post-natal. Should the authors not looked at the day of onset of symptoms since if onset is very early, it seems unlikely transmission was post-partum? It would be exceedingly unlikely that HSV-2 was acquired from post-partum transmission, especially from a visiting relative.\n\nIs it possible to say how many of the deaths were attributable to HSV?\n\nMild, moderate and severe damage need to be defined in Methods.\n\nHow can one have a developmental delay at one month of age? An infant does not have to even smile to be normal at one month of age.\n\nDiscussion – “Future investigations of the national picture with respect to this rare but devastating condition should consider the role of postnatal acquisition and virus type, and the relationship between pre-term delivery and neonatal HSV following both primary and recurrent maternal genital infection.” – This is a rather vague sentence. How will investigating the virus type and the role of preterm delivery help in the future?\n\nDiscussion – “The introduction of screening for HSV susceptibility in pregnancy would not address the issue of infection in pre-term babies exposed to recurrent infection, in view of the lack of transfer of maternal antibody.” – Those who believe in identifying susceptible women advise that they practice safe sex if their partner is positive. I could not understand how this relates to preterm infants.\n\nDiscussion – “The important contribution of postnatal acquisition must not be ignored or minimized in the debate around pregnancy screening. The assumption that neonatal herpes is mainly due to maternal genital infection must be challenged – in this study almost a quarter of all cases, and about half of those where a likely source was identified, were attributable to postnatal exposure to cold sores and other herpetic lesions.” As mentioned above, the authors have not convincingly shown that a large number of cases are acquired after birth.\n\nDo the authors truly believe that warning all new parents about this rare condition is a good idea and would prevent cases?\n\nDiscussion – “Nonetheless, the overall proportion of adverse outcomes was lower than in the earlier study (overall 51% compared with 58%).” Here and throughout the paper, the authors should not imply that there is a difference unless it is statistically significant.\n\nMinor points:\nIt would be helpful to the reader if both absolute numbers and percentages are provided in the abstract and throughout the paper.\n\nEthical statement - “Between 2004 and 2006” would really only be 2005. How about “2004 through 2006”?\n\nIt is not clear why the data are being submitted for publication about a decade after the last follow-up data were collected.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "98091",
"date": "25 Nov 2021",
"name": "Justin C Konje",
"expertise": [
"Reviewer Expertise 1. Fetal growth 2. Perinatal infections"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis important surveillance study for the period 2004-6 was the second in the British Isles, the first was for the period 1986-91. The key findings were an increased incidence (almost doubled that of the previous period), a higher prevalence in preterm infants and 57% secondary to HSV-2. The authors suggest that improvements in diagnostic techniques and clinical awareness may have had an impact on the reported incidence. I believe that this surveillance report adds to our continuing drive to increase not only awareness but the importance for clinicians to recognise that neonatal HSV remains an important cause of morbidity and mortality.\nMajor Comments:\nAbstract:\nIt reads as if this was a comparative study – making repeated reference to the surveillance of 1986-1991 in the results paragraph. While this is important it should not be in the results. Reference to this is appropriate in the conclusion.\n\nThe comment that “the increased of adverse outcomes associated with HSV-2 in the earlier study was confirmed and strengthened” should be revised and I will suggest deleting the word “strengthened” as there is no evidence to back this up.\nIntroduction\nAlthough prior HSV-1 offers more relative protection to HSV-2 infection, there are some studies that also show that prior HSV-2 offers some protection against HSV-1 (not to the same degree) – e.g. Brown ZA et al. (1997)1; Nahmais et al. (1990)2.\n\nMethods\nIn the case identification, reference is made to paediatricians reporting on stillbirths with confirmed HSV infection. This is unlikely to be reported by paediatricians. If this is indeed the case, then it is possible that some data would be inaccurate. Ideally this should come from the laboratory where the diagnosis is confirmed. Perhaps this should be discussed as a limitation of this approach. Furthermore, this is a survey of neonatal HSV and stillbirths should not be included as neonates. Furthermore, the Flow chart rightly does not include the SBs – so exclude please.\n\nProvide a definition of ‘mild moderate or severe disease”\nResults\nIn the maternal and birth factors, there were 29 emergency CS and 2 elective CS. An important factor in vertical transmission at the time of delivery is pre-delivery rupture of membranes. Was it possible to obtain this information? This is very important with respect to understanding of how these 26 neonates acquired HSV. This is also most likely linked to the gestational age. It would be interesting to know how many of the CS were <36 weeks.\n\nWith regards to the timing of symptoms –it was not immediately clear to me whether reference to initiating treatment was also related to the onset of symptoms. Important information that would provide clues to the time of acquisition would be the time of onset of symptoms from birth. The incubation period would help identify those infections acquired at the time of delivery and those acquired after birth.\nDiscussion\nIs there any evidence from the data that over the period of the surveillance, the reported incidence was increasing? If there is none, then the statement on improvements in diagnostic techniques is unlikely to have been a significant factor in the risen incidence. I agree that increased awareness on the part of clinicians is most likely to have been a major driver of the higher reported number of cases.\n\nAt the end of the third paragraph the authors state that the increased incidence in preterm babies may be because of lack of adequate transfer of antibodies following perinatal exposure to primary or recurrent infection. I would agree with primary but not recurrent infections. I am more inclined to believe that several factors are at play – premature rupture of membranes especially preterm (which increased vertical transmission) and genital co-infections that are known to be a factor in preterm delivery – all increasing the risk of vertical transmission.\n\nThere is no evidence that maternal transfer of antibodies is influenced by gestational age. It is influenced by timing of infection/duration of infection prior to delivery. Please revise this statement in paragraph 5.\n\nI am not sure that enough evidence is presented to challenge the fact that more neonatal HSV is acquired genitally (paragraph 5). I think the authors ought to discuss prelabour rupture of fetal membranes as a factor that increases risk and while this may not have been collected, it must be discussed as a major weakness of the study (i.e. inference on timing of infection cannot be made without this).\n\nI am not sure how warning all parents would prevent cases – I would suggest that better education of clinicians (family practitioners and paediatricians) would increase case identification and institution of early treatment\nMinor\nData are plural.\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? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-163
|
https://f1000research.com/articles/9-160/v1
|
03 Mar 20
|
{
"type": "Research Article",
"title": "Income and obesity in an urban poor community: a cross-sectional study",
"authors": [
"Jo Ann Andoy-Galvan",
"Halyna Lugova",
"Sapna S. Patil",
"Yin How Wong",
"Gul M. Baloch",
"Adlina Suleiman",
"Rusli Nordin",
"Karuthan Chinna",
"Halyna Lugova",
"Sapna S. Patil",
"Yin How Wong",
"Gul M. Baloch",
"Adlina Suleiman",
"Rusli Nordin",
"Karuthan Chinna"
],
"abstract": "Background: Recent studies have shown that higher income is associated with a higher risk for subsequent obesity in low- and middle-income countries, while in high-income countries there is a reversal of the association – higher-income individuals have a lower risk of obesity. The concept of being able to afford to overeat is no longer a predictor of obesity in developed countries. In Malaysia, a trend has been observed that the prevalence of obesity increases with an increase in income among the low-income (B40) group. This trend, however, was not further investigated. Therefore, this study was performed to investigate the association of income and other sociodemographic factors with obesity among residents within the B40 income group in an urban community. Methods: This cross-sectional study used a systematic sampling technique to recruit participants residing in a Program Perumahan Rakyat (PPR), Kuala Lumpur, Malaysia. The sociodemographic characteristics were investigated through face-to-face interviews. Weight and height were measured, and body mass index (BMI) was calculated and coded as underweight, normal, overweight and obese according to the cut-off points for the Asian population. A chi-squared test was used to compare the prevalence of obesity in this study with the national prevalence. A generalized linear model was introduced to identify BMI predictors. Results: Among the 341 participants, 25 (7.3%) were underweight, 94 (27.6%) had normal weight, 87 (25.5%) were overweight, and 135 (39.6%) were obese. The proportion of obese adults (45.8%) was significantly higher than the national prevalence of 30.6% (p<0.001). Among all the tested variables, only income was significantly associated with BMI (p=0.046). Conclusion: The proportion of obesity in this urban poor community was higher compared with the national average. BMI increased as the average monthly household income decreased.",
"keywords": [
"obesity",
"prevalence",
"risk factors",
"socio-economic status",
"income"
],
"content": "Introduction\n\nObesity prevalence has tripled since the 1970s. Currently, it has reached epidemic proportions throughout the globe and become a significant cause of morbidity and mortality (WHO, 2020). It is a major risk factor in the development of the leading causes of global deaths, such as diabetes, cardiovascular diseases and cancer (Al-Goblan et al., 2014; De Pergola & Silvestris, 2013; Hubert et al., 1983).\n\nMalaysia is the most obese country among the Southeast Asian nations (Ng et al., 2014). Two out of three Malaysians are overweight or obese (Aris et al., 2015). Large-scale obesity studies in the country showed a two-fold increase in the prevalence of people categorised as overweight, from 16.6% to 30%, and a four-fold increase in obesity prevalence, from 4.5% to 17.7%, in the last two decades (Aris et al., 2015; Lin et al., 2006). The main driver of obesity is an imbalance between energy consumption and expenditure (Romieu et al., 2017). However, our choices of eating and physical exercise are influenced by multiple factors and the concept of an obesogenic environment has been a topic of interest lately, that is, an environment that increases our risk of gaining weight and becoming obese (Lipek et al., 2015). Recent studies have shown complexity in the association of income and obesity: a higher income is associated with a higher risk of subsequent obesity in low- and middle income countries, but in high-income countries the reverse is observed, where those with a higher income are less likely to be obese (McLaren, 2007). The concept that richer people can afford to overeat may no longer be true among well-developed nations. The reversal hypothesis was systematically tested using individual- and aggregate-level data for 67 nations representing all regions of the world and findings were consistent with individual studies in different countries: the influence of socioeconomic status on obesity shifts from positive to negative with national income (Pampel et al., 2012).\n\nAccording to income classification in Malaysia, the income groups B40, M40 and T20 represent the lowest 40%, middle 40%, and highest 20% of incomes, respectively, among the country’s population. A national large-scale obesity study in 2015 revealed a positive trend among the low-income (B40) group but a mixed trend among the middle- and high-income groups (Aris et al., 2015). Also worth noting is the high prevalence rate of obesity in states with the highest (Putrajaya) and lowest urbanization (Perlis) levels (Aris et al., 2015; Department of Statistics, Malaysia, 2010). Nevertheless, the observed trend may not be universally applicable across heterogenous rural and urban community settings, where pockets of poverty are often found.\n\nThe inconsistencies with regards to the positive and negative associations of income with obesity might be due to a lack of studies among poor communities. These communities might differ in obesity prevalence along with its associated factors, and there are no current studies that explore this phenomenon. Therefore, this study was performed to investigate the association of income and other sociodemographic factors associated with obesity among residents within the B40 income group in an urban community.\n\n\nMethods\n\nThis cross-sectional study was carried out among the residents of the low-cost high-rise flats of a Community Housing Program, or PPR (Program Perumahan Rakyat in local language) in Kuala Lumpur. PPRs have been developed by the National Housing Department (Jabatan Perumahan Negara or JPN) since the 1998 in an effort to provide affordable housing to low socioeconomic groups (Goh et al., 2011). A group of 150 medical students were deployed to collect data from all the 21 floors of the two residential building blocks during weekends of February 2019 and August 2019.\n\nThe sample size (n) was calculated using the Krejcie and Morgan formula for prevalence studies of a known population (Krejcie & Morgan, 1970). Prevalence used in the formula was the current prevalence of people who are overweight and obese for both genders, which is close to 50% (Aris et al., 2015). Out of 4229 residents, a total of 380 residents were expected to participate in the survey. Residents were approached at their home by data collectors using a systematic sampling procedure to ensure even representation of units from all the 21 floors. Data collectors were divided into 20 groups, each containing 7–8 collectors, 10 of which were assigned to building block A and 10 of which were assigned to building block B. Each group was assigned 2–3 floors of the 21-floor building block and were instructed to carry out systematic random sampling on their floors. If residents of one house did not consent, the data collectors moved to the next house. Within the selected units, all the residents were invited for an interview. The inclusion criteria included: (1) residents of the PPR; (2) Malaysian citizens; (3) aged =/>5 years old. Physically disabled/bedridden individuals were excluded (Figure 1).\n\nSociodemographic data, including age, gender, race, educational level, marital status, household income and occupation, were collected by interviewing the residents using a questionnaire, and then their weight and height were measured to calculate their body mass index (BMI). Interviews were carried out by the data collectors; third year and fourth year medical students who had been trained to use the questionnaire. These data collectors had no prior interaction with the participants, but researchers regularly visit the community as part of ongoing research at our university. Interviews took place in the participants’ homes and lasted 30–45 minutes.\n\nBody weight of the participants was measured in light clothing without footwear using Omron HBF-375 Body Composition Monitor digital weighing scale (Omron, Petaling Jaya, Malaysia). Height was measured without shoes using a measuring tape to the nearest 0.1 cm. BMI of each participant was calculated using a standard formula of weight (kg) divided by the square of the height (m2) in kg/m2.\n\nThe participants were categorized based on the classification of the 2004 Malaysian Clinical Practice Guidelines (CPG) on Management of Obesity (Ismail et al., 2004) which has adopted the recommended cut off points for the Asian population (Nishida et al., 2004). Under these guidelines, BMI was classified into six categories: underweight (<18.50 kg/m2), normal (18.50 – 22.99 kg/m2), overweight (23.00 – 27.49 kg/m2), obese I (27.50 – 34.99 kg/m2), obese II (35.00 – 39.99) and obese III (>40 kg/m2) (Zainudin et al., 2011). For this study, we combined the obese I, II and III into one category of “obese”. For comparison with the national data, the BMI was also categorized according to the WHO Classification. (Aris et al., 2015).\n\nFor children aged 5 to 17 years, the BMI-for-age percentile based on Center for Disease Control growth charts, which are gender-specific, was used to categorize BMI (Kuczmarski et al., 2002).\n\nData were analysed using IBM Statistical Package for Social Sciences version 23 (IBM, New York, USA). Frequencies, means, standard deviations were used to summarize the findings. Chi square was used to compare the obesity prevalence of the sample group with the national prevalence. Household income was skewed to the right; hence, it was log transformed. All the predictor variables were tested using multivariate analysis to determine the association with BMI. We used an alpha level of 0.05 for all the statistical tests.\n\nThis study received Institutional Review Board approval from Taylor's University Center for Research Management (HEC 2019/058). The study was conducted according to the Declaration of Helsinki. Prior written informed consent was obtained from all participants for participation and publication of data.\n\n\nResults\n\nThere was a total of 380 participants, of which 341 completed anthropometric measurements. Of these, there was a total of 328 respondents, including 261 adults and 80 children (aged below 18 years old) from 209 household units, who provided full sociodemographic information (Galvan, 2020). Table 1 shows the sociodemographic characteristics of the 328 participants. Among the 261 adult participants that completed anthropometric measurements, 29.1% and 45.6% were overweight and obese, respectively (Table 2). Among the respondents aged below 18 years, the prevalence of an overweight and obese BMI was 13.8 % and 20%, respectively (Table 3).\n\nBMI, body mass index; RM, Malaysian ringgit.\n\naCPG, Clinical Practice Guidelines in Malaysia bWHO, World Health Organization. BMI, body mass index.\n\nBMI, body mass index.\n\nAs shown in Table 4, the prevalence of obesity in our sample was significantly higher as compared to the average national prevalence by using both CPG classification of BMI (45.6% vs. 30.6%, p<0.001) and WHO classification of BMI (29.9% vs. 17,7%, p<0.001). The prevalence of obesity in this study was similar to that observed in the federal territory of Putrajaya (43%), which has the highest obesity prevalence in the country (Aris et al., 2015).\n\nBMI, body mass index; CPG, Clinical Practice Guidelines in Malaysia; WHO, World Health Organization.\n\nA generalized linear model was used to test the association between the predictor variables and BMI. As shown in Table 5, among all the tested variables, gender and income were significantly associated with BMI. Participants with a higher income had a lower BMI (b -1.86; p value 0.046). Males had a lower BMI than females (b-1.475; p value 0.037).\n\nb (SE)*\n\nBMI, body mass index.\n\n\nDiscussion\n\nThe obesity prevalence in this study was significantly higher compared to the national prevalence (Aris et al., 2015). Contrary to the results from the national survey (Aris et al., 2015), income was negatively associated with BMI after controlling for other variables in this low-income (B40) population group; in other words, the individuals with a higher income had a lower BMI. The negative association was similar to that of high-income countries, where a socioeconomically developed environment may possibly contribute to the reversal (Pampel et al., 2012). In contrast to the built environment in rural areas, families with a higher income among this community may be able to afford accessible healthy food choices along with various available weight loss programs, walkable entertainment centres, shopping malls and healthcare facilities (Ellis et al., 2016); thus, they are less obese as compared to the rest of the community. This supportive environment decreases the risk of obesity for urban dwellers, while the concept of being able to afford to overeat may still be true among wealthy rural dwellers (Creatore et al., 2016). Our results confirm the arguments discussed in the review of 2,009 population-based studies done by the NCD Risk Factor Collaboration (Bixby et al., 2019). The researchers found that more than 55% of the rise in global obesity was from rural areas. Their findings negate the assumptions that urbanization was the main driver of the obesity epidemic. Assumptions of sedentary lifestyles with access to processed food in urban places, and the general preconceived notion of manual labor and healthy eating from one’s own garden among rural dwellers may not be true. Automation has influenced our agricultural activities and distribution or shipment of processed food to rural areas. (Bixby et al., 2019) In the national survey done by Aris et al. (2015), both the states with highest and lowest urbanization levels had high prevalence rates of obesity. Putrajaya, with 100% urbanization, reported a 43% rate of obesity and Perlis, with 51.4% urbanization, reported a rate of 36% rate of obesity, ranking 2nd together with the state of Melaka. This report contradicts the general idea that obesity applies to urban communities and malnutrition to rural communities. In our report, this urban poor community has a greater prevalence of obesity compared to the general population but the distribution of obesity among them was skewed towards those with a low income. The driver could possibly be that the advantages of the available supportive environment, such as gyms and healthy food choices, in the urban-built environment are missed by those with a lower income.\n\n\nConclusion\n\nThe obesity rate in this urban poor community is high. The monthly household income is negatively associated with obesity; people with a higher income have a lower BMI.\n\nTo understand further the complexity of the relationship between income and obesity, we recommend further investigations involving more than one PPR community. Determining monthly food and health expenses of the families will increase the precision of information on the household income level. Additionally, a wider study could compare trends between urban and rural poor communities.\n\n\nLimitations\n\nOur findings are limited to one community, and the cross-sectional nature of this study does not permit any temporal relationships to be deduced. Monthly household income disclosed to the interviewers may not reflect the actual figures; the residents in this community are recipients of the government housing project and therefore represent the poorest 40% of the population and should receive a salary of ~3,000 ringgit or below. However, salaries higher than 3,000 ringgit were reported in this study.\n\n\nData availability\n\nHarvard Dataverse: Income and obesity in an urban poor community: a cross-sectional study. https://doi.org/10.7910/DVN/XRO2PI (Galvan, 2020)\n\nThis project contains the following underlying data:\n\n- Adults_22236F.tab (BMI data for adults in the study)\n\n- Children_22236.tab (demographic and BMI data for children in the study)\n\n- Combined_Data_22236F.ods (demographic and BMI data for all participants in the study)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nHousehold income data are not included in the dataset but will be available upon request from the corresponding author. This is to protect the confidentiality of the income received by the residents living in a housing project intended for the poorest families in the country. There are numerous determinants of salary, which lead to diverse income figures among the residents and this should not be misinterpreted by taking the information on the income separately. Researchers who would like to acquire the data for the purpose of statistics may write to the corresponding author (JoAnnAndoy.Galvan@taylors.edu.my) stating reasons for the request.",
"appendix": "Acknowledgements\n\nThe authors would like to thank the following students who helped in the data collection: Ooi Tzu Chia, Saung Thinzar, Navina Nagarajan, Shin Chen Keng, Wong Siew Ern, Deevetra Roslyn, Tsepo Anthony Phori, Wong Joon Leek and the Universiti Pertahanan Nasional Malaysia School of Medicine Year 3, Batch 7 and Year 4, Batch 6 students.\n\nThis manuscript is not submitted nor being considered for publication elsewhere but has been partly presented and won the best oral presentation under the “Session Health Promotion, Health Education, Obesity and Health’ in the 5th International Conference in Public Health, held in Kuala Lumpur, Malaysia, 2019.\n\n\nReferences\n\nAris HT, Yusoff MF, Ghani AA, et al.: National Health and Morbidity Survey. 2015; [cited 2019 July 18]. Reference Source\n\nAl-Goblan AS, Al-Alfi M, Khan MS: Mechanism linking diabetes mellitus and obesity. Diabetes Metab Syndr Obes. 2014; 7: 587–591. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBixby H, Bentham J, Zhou B, et al.: Rising rural body-mass index is the main driver of the global obesity epidemic in adults. Nature. 2019; 569(7755): 260–264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCreatore MI, Glazier RH, Moineddin R, et al.: Association of Neighborhood Walkability With Change in Overweight, Obesity, and Diabetes. JAMA. 2016; 315(20): 2211–20. PubMed Abstract | Publisher Full Text\n\nDe Pergola G, Silvestris F: Obesity as a major risk factor for cancer. J Obes. 2013; 2013: 291546. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEllis G, Hunter R, Tully M, et al.: Connectivity and physical activity: Using footpath networks to measure the walkability of built environments. Environ Plan B. 2016; 43(1): 130–151. Publisher Full Text\n\nGalvan JA: Income and obesity in an urban poor community: a cross-sectional study. Harvard Dataverse, V4, UNF:6:bXkGg+VkQHPrPMvi8gsaEw== [fileUNF]. 2020. Publisher Full Text\n\nGoh A, Tee A, Yahaya: Public Low-Cost Housing in Malaysia: Case Studies on PPR Low-Cost Flats in Kuala Lumpur. Journal of Design and the Built Environment. 2011; 8. Reference Source\n\nHubert HM, Feinleib M, McNamara P, et al.: Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham Heart Study. Circulation. 1983; 67(5): 968–977. PubMed Abstract | Publisher Full Text\n\nIsmail IS, Bebakar M, Noor I, et al.: Clinical Practice Guidelines on Management of Obesity. 2004; [cited 2020 Feb 05]. Reference Source\n\nKrejcie RV, Morgan DW: Determining sample size for research activities. Educational & Psychological Measurement. 1970; 30: 607–610. Publisher Full Text\n\nKuczmarski RJ, Ogden CL, Guo SS, et al.: 2000 CDC Growth Charts for the United States: methods and development. Vital Health Stat 11. 2002; 11(246): 1–190. PubMed Abstract\n\nLin K, Mohamad N, Rasat R, et al.: The Third National Health and Morbidity Survey 2006 (NHMS III) Nutritional Status. 2006; [cited 2019 July 18]. Reference Source\n\nLipek T, Igel U, Gausche R, et al.: Obesogenic environments: environmental approaches to obesity prevention. J Pediatr Endocrinol Metab. 2015; 28(5–6): 485–495. PubMed Abstract | Publisher Full Text\n\nMcLaren L: Socioeconomic status and obesity. Epidemiol Rev. 2007; 29: 29–48. PubMed Abstract | Publisher Full Text\n\nNg M, Fleming T, Robinson M, et al.: Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014; 384(9945): 766–781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNishida C, Barba C, Cavalli-Sforza T, et al.: Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004; 363(9403): 157–63. PubMed Abstract | Publisher Full Text\n\nPampel FC, Denney JT, Krueger PM: Obesity, SES, and economic development: a test of the reversal hypothesis. Soc Sci Med. 2012; 74(7): 1073–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPopulation Distribution and Basic Demographic Characteristic Report. Department of Statistics Malaysia, 2010. Reference Source\n\nRomieu I, Dossus L, Barquera S, et al.: Energy balance and obesity: what are the main drivers? Cancer Causes Control. 2017; 28(3): 247–258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Obesity. 2020. Accessed: February 27, 2020. Reference Source\n\nZainudin S, Daud Z, Mohamad M, et al.: A Summary of the Malaysian Clinical Practice Guidelines on Management of Obesity 2004. Journal of the ASEAN Federation of Endocrine Societies. 2011; 26: 101–104. Publisher Full Text"
}
|
[
{
"id": "61436",
"date": "31 Mar 2020",
"name": "Gajanan D Velhal",
"expertise": [
"Reviewer Expertise Nutrition",
"Epidemiology of Communicable and Non communicable diseases",
"Epidemiological studies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nContrary to the existing belief that Obesity is more common among the people belonging to higher socioeconomic status, present study reveals that prevalence of obesity is seen more among individuals with low income categories, whose economic status is improving. This clearly indicates the need to focus on the selected category of people to influence overall prevalence of obesity in the community. Reasons to exclude physically handicapped and bed ridden patients is not clear. It would have been better to give elaborate description on employment of systematic random sampling technique for selection of study subjects.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "62102",
"date": "23 Apr 2020",
"name": "Khalid Abdul Kadir",
"expertise": [
"Reviewer Expertise Diabetes",
"obesity and metabolic diseases",
"epidemiology of diabetes and metabolic diseases",
"clinical trials on diabetes",
"animal studies on diabetes and metabolic syndrome"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 cross sectional study of the prevalence obesity and overweight amongst the lower 40% of a Malaysian population living in an urban government housing scheme. The methodology used was very appropriate a properly calculated sample size for the population, and using systemic sampling procedure with face to face interviews etc.\nThe criteria used for overweight and obesity was also appropriate for the Asian/Malaysian population The data obtained included socio demographic data including income There was no data about the diet or food calorie intake nor of energy expenditure ie physical work, exercise etc The data obtained was interesting and novel in that it showed increased prevalence of obesity/overweight in the lower income group but the prevalence is less in this group if the income is higher. The prevalence is then compared to a recent nationwide prevalence survey by Aris et al. and showed higher prevalence compared to national average. The findings would have been improved if they had looked at the racial group involved compared to the national data, and to include the nutritional and dietary habits\nOverall a short interesting paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-160
|
https://f1000research.com/articles/8-166/v1
|
07 Feb 19
|
{
"type": "Research Article",
"title": "Accuracy of linear measurements obtained from stitched cone beam computed tomography images versus direct skull measurements",
"authors": [
"Doaa Ahmed Fouad Hamed",
"Reham Mohamed Hamdy",
"Sahar Hosny El Dessouky",
"Reham Mohamed Hamdy",
"Sahar Hosny El Dessouky"
],
"abstract": "Background: To assess whether the linear measurements obtained from stitched cone beam computed tomography (CBCT) images were as accurate as the direct skull measurements. Methods: Nine dry human skulls were marked with gutta-percha at reference points to obtain Twenty-two linear measurements on each skull. Ten measurements in the cranio-caudal plane, two measurements in the antero-posterior plane, and ten measurements in the medio-lateral plane. CBCT linear measurements obtained using stitching software were measured and compared with direct skull measurements. Results: The absolute Dahlberg error between direct linear measurements and linear measurements on stitched CBCT images ranged from (0.07 mm to 0.41 mm). The relative Dahlberg error ranged from (0.2% to 1.8%). Moreover, Intra-class Correlation Coefficient (ICC) ranged from (0.97 to 1.0) indicating excellent agreement. Conclusion: Stitched CBCT linear measurements were highly comparable to the direct skull measurements using a digital caliper.",
"keywords": [
"Cone beam CT",
"linear measurements",
"accuracy",
"direct measurements",
"field of view",
"stitched images."
],
"content": "Introduction\n\nThe use of cone beam computed tomography (CBCT) machines in dentistry started in the second half of the 1990s1. Now, CBCT is extensively used in the dental field for implant planning, in endodontics, maxillofacial surgeries and orthodontics2.\n\nIn the field of orthodontics, analysis of cephalometric radiographs requires accurate identification of specific landmarks for precise measurements between these landmarks3. As a consequence, the small field of view (FOV) CBCT systems available in small clinics cannot yet satisfy the needs of maxillofacial surgeons or orthodontists4. Thus, visualizing all of these landmarks on the same scan is not always possible5.\n\nIn order to compensate for this shortcoming, small FOV images can be scanned and then fused together to produce a single large FOV image. However, there are few data to show whether this fused image is as precise as a single image of the whole area of interest4,6.\n\nTherefore, the aim of the current study was to assess the diagnostic accuracy of stitched CBCT linear measurements versus direct measurements on skulls.\n\n\nMethods\n\nThe current study was conducted on nine dry human skulls obtained from the Anatomy department, Faculty of Medicine, Cairo University to avoid the exposure of living humans to unnecessary radiation doses. 26 anatomical landmarks were identified on each skull (Table 1). Gutta percha cones (GE16121542, META BIOMED) were glued and used as radiopaque markers (Figure 1–Figure 3).\n\n22 linear measurements were taken and recorded using a high precision sliding digital caliper (6400192, Allendale Electronics Ltd, Hertfordshire, UK). Ten measurements in the cranio-caudal plane (Table 2), two measurements in the antero-posterior plane (Table 3), and ten measurements in the medio-lateral plane (Table 4). 22 direct linear measurements were measured and were considered to be the gold standard in the study (Figure 4–Figure 6).\n\nR – right, L - left\n\nFor soft tissue simulation, the skulls were covered with 20 layers of pink modelling wax (1mm thick each) (Tenatex eco, Kemdent) to achieve an average of 14–16 mm wax thickness7.\n\nThe skulls were stabilized in the Planmeca ProMax 3D Mid CBCT machine using a wooden stand passing through the foramen magnum and were oriented using the laser beams (Figure 7). Three consecutive FOVs were scanned: two scans each of the size 160 ×60 mm (single arch) for the mandible and the maxilla separately (Figure 8 and Figure 9) and one scan with a FOV size 200 ×100 mm for the upper third of the face (Figure 10). Each one of the three FOVs was scanned separately using a voxel resolution 0.2 mm, 90 kVp and 10 mA, then stitching was performed using Romexis software (Planmeca Romexis Viewer Launcher 4.5.0.R) creating one large volume (Figure 11). After completion of the stitching procedure the linear measurements were obtained from the stitched CBCT images for a later comparison with the gold standard (Figure 12 and Figure 13).\n\nStatistical analysis was performed on SPSS (version 17). For assessment of the agreement between all measurements with the reference method, Dahelberg error (DE), and Relative Dahelberg Error (RDE) were used together with Intra-class Correlation Coefficients (ICC) including the 95% confidence limits of the coefficient calculated assuming analysis of variance two-way mixed model ANOVA with absolute agreement on SPSS. To measure and quantify the size of the differences, Bland and Altman 95% confidence Limits of Agreements (LOA) were applied.\n\n\nResults\n\nThe results of the current study showed that, the difference between the mean of the direct linear measurements and the mean of the linear measurements conducted on the stitched CBCT images ranged from (-0.25 mm to 0.5 mm), the positive and negative values indicating that there was no obvious pattern of over or underestimation in the stitched CBCT measurements.\n\nN – Nasion, ANS - Anterior nasal spine, PNS - Posterior nasal spine, A – A point, B – B point, Me – Menton, ZYF - Zygomatic foramen, Co – Condyle, GO – Mandibular gonion, MOR – Medial orbital wall, LOR – Lateral orbital wall, ORF – infra-orbital foramen, GP – Greater palatine foramen, MF – Mental foramen, AR – Anterior ramus, PR – Posterior ramus, R – right, L - left\n\nMean absolute difference of all measurements was (0.11± 0.12 mm). Bland and Altman Lower limit of agreement ranged from (-0.92 mm to -0.06 mm). Bland and Altman Upper limit of agreement ranged from (0.1 mm to 1.24 mm).\n\nThe absolute Dahlberg error between direct linear measurements and linear measurements on stitched CBCT images ranged from (0.07 mm to 0.41 mm). The relative Dahlberg error ranged from (0.2% to 1.8%). Moreover, Intra-class Correlation Coefficient (ICC) ranged from (0.97 to 1.0). (Table 5).) indicating excellent agreement.\n\n\nDiscussion\n\nThe smaller the scan FOV, the higher the spatial resolution of the image8. Stitching of small CBCT images to create a large image can be very useful to collect the needed cranio-maxillofacial data with small FOV machines9. Increasing the FOV can be done by automatically fusing up to three small FOVs to obtain a larger FOV10.\n\nCBCT ‘‘Stitching’’ option could be useful but whether it is precise enough to obtain accurate and reliable measurements remains doubtful11. Assessing the accuracy of stitched CBCT measurements is infrequently mentioned in current literature, as in the studies conducted by Kopp and Ottl; 2010, Kim et al.; 2012, Egbert et al.; 2015, and Srimawong et al.; 20155,8,10,12.\n\nThe results of the current study showed that the relative Dahlberg error ranged from 0.2% to a maximum of 1.8%. Consequently, the error was considered small and clinically non-significant as the measurement error in craniofacial imaging is considered clinically acceptable up till the value of 5%13,14.\n\nThe results of the current study go in agreement with the study performed by Srimawong et al.; 2015 on 10 dry human mandibles12. Their results showed that, the mean absolute differences between direct measurements and stitched CBCT measurements for vertical and horizontal distances were (0.27±0.24 mm) and (0.34±0.27 mm), respectively. Their results showed that the stitched CBCT measurements were highly accurate comparable to the direct measurements.\n\nIn support of the present results, Egbert et al.; 20158 research revealed that the mean difference between the direct linear measurements and the stitched CBCT measurements was 0.34 mm with a 95% confidence interval of (0.24 to 0.44 mm). They concluded that the precision of stitched CBCT measurements allow accurate construction of implant surgical stents.\n\nMoreover, the results of Kopp and Ottl; 201010 further agree with those obtained from the current study. They used an automated method to increase the FOV of CBCT images by stitching three small FOV volumes to obtain a larger FOV one. They concluded that, the stitching software was accurate in the obtained linear measurements.\n\nOn the same line of agreement, a study was performed by Kim et al.; 20125 to investigate whether images of skulls obtained by both manual and automatic stitching of three CBCT images, provided accurate measurements as those obtained by multidetector computed tomography (MDCT). The results showed that the mean difference between automatically stitched CBCT images and the reference images ranged from (-0.8944 mm to -1.0628 mm).\n\n\nConclusion\n\nStitched CBCT linear measurements were highly comparable to the direct skull measurements. However, a percent of error should be expected from CBCT-derived measurements.\n\n\nData availability\n\nUnderlying data is available from Open Science Framework\n\nOSF: Dataset 1. Accuracy of Linear Measurements Obtained from Stitched Cone Beam CT Images Versus Direct Skull Measurements https://doi.org/10.17605/OSF.IO/SUTWK15",
"appendix": "Grant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPauwels R, Araki K, Siewerdsen JH, et al.: Technical aspects of dental CBCT: state of the art. Dentomaxillofac Radiol. 2015; 44(1): 20140224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlamri HM, Sadrameli M, Alshalhoob MA, et al.: Applications of CBCT in dental practice: a review of the literature. Gen Dent. 2012; 60(5): 390–400; quiz 401–2. PubMed Abstract\n\nHatcher DC: Operational principles for cone-beam computed tomography. J Am Dent Assoc. 2010; 141 Suppl 3: 3S–6S. PubMed Abstract | Publisher Full Text\n\nGiesel FL, Mehndiratta A, Locklin J, et al.: Image fusion using CT, MRI and PET for treatment planning, navigation and follow up in percutaneous RFA. Exp Oncol. 2009; 31(2): 106–114. PubMed Abstract | Free Full Text\n\nKim MK, Kang SH, Lee EH, et al.: Accuracy and validity of stitching sectional cone beam computed tomographic images. J Craniofac Surg. 2012; 23(4): 1071–1076. PubMed Abstract | Publisher Full Text\n\nBrüllmann D, Seelge M, Schömer E, et al.: Alignment of cone beam computed tomography data using intra-oral fiducial markers. Comput Med Imaging Graph. 2010; 34(7): 543–552. PubMed Abstract | Publisher Full Text\n\nSchropp L, Alyass NS, Wenzel A, et al.: Validity of wax and acrylic as soft-tissue simulation materials used in in vitro radiographic studies. Dentomaxillofac Radiol. 2012; 41(8): 686–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEgbert N, Cagna DR, Ahuja S, et al.: Accuracy and reliability of stitched cone-beam computed tomography images. Imaging Sci Dent. 2015; 45(1): 41–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaidi H, Montandon ML, Alavi A: The clinical role of fusion imaging using PET, CT, and MR imaging. Magn Reson Imaging Clin N Am. 2010; 18(1): 133–[149] . PubMed Abstract | Publisher Full Text\n\nKopp S, Ottl P: Dimensional stability in composite cone beam computed tomography. Dentomaxillofac Radiol. 2010; 39(8): 512–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaoumova J, Lindman R: A comparison of manual traced images and corresponding scanned radiographs digitally traced. Eur J Orthod. 2009; 31(3): 247–253. PubMed Abstract | Publisher Full Text\n\nSrimawong P, Krisanachinda A, Chindasombatjaroen J: Accuracy of linear measurements in stitched versus non-stitched cone beam CT images. CU Dent J. 2015; 38: 93–104. Reference Source\n\nHilgers ML, Scarfe WC, Scheetz JP, et al.: Accuracy of linear temporomandibular joint measurements with cone beam computed tomography and digital cephalometric radiography. Am J Orthod Dentofacial Orthop. 2005; 128(6): 803–811. PubMed Abstract | Publisher Full Text\n\nTarazona-Álvarez P, Romero-Millán J, Peñarrocha-Oltra D, et al.: Comparative study of mandibular linear measurements obtained by cone beam computed tomography and digital calipers. J Clin Exp Dent. 2014; 6(3): e271–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamed D, Hamdy RM, Dessouky SHE: Accuracy of Linear Measurements Obtained from Stitched Cone Beam CT Images Versus Direct Skull Measurements. 2019. http://www.doi.org/10.17605/OSF.IO/SUTWK"
}
|
[
{
"id": "52211",
"date": "12 Aug 2019",
"name": "Luca Fiorillo",
"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 study is very interesting, and having assessed the reliability of radiographic examinations is very useful for clinical practice.\nWe should only understand, as a future perspective, the differences between the different devices used, or between the different exposure settings; it could be mentioned in \"Discussion\" section as a limitation of the study.\nThe work has been well done, and the results are in agreement with other studies in the literature.\nIn the \"Keywords\" section, I recommend using Meshwords (https://meshb.nlm.nih.gov/)\n\nI suggest to expand the \"Introduction\" section: Add notes on the history of radiology in maxillofacial and oral medicine12. Add clinical notes on the use of three-dimensional radiographic examinations, and how these may influence rehabilitation3. Furthermore it is necessary to subdivide the section into two sub-sections, for example \"Background\" and \"Aim\".\n\nThe \"Methods\" section should be implemented only by improving image quality if possible.\n\nIn the \"Conclusions\" section, cite a future perspective of the study, and the function of this research in clinical practice.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "55480",
"date": "13 Nov 2019",
"name": "Pathawee Khongkhunthian",
"expertise": [
"Reviewer Expertise dental implantology",
"biomaterial in tissue regenneration."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe comparative study between direct measurement of the skull and the stitching CBCT data obtained from different view had been well designed and performed. The statistical analysis are appropriate and sufficient to make the conclusion. The study showed the accuracy of stitching method using a commercial software.\nSome of the issues should be raised in the discussion part for more information of the study.\nThe use of dry skull compared to the real clinical situation in term of soft tissue, does the soft tissue impair the clinical accuracy?\n\nIf comparing the measurements between the stitching technique to the large field of view in CBCT (full skull sensor), how is the accuracy?\n\nThe limitation of the study should be mentioned.\n\nThe clinical implication?\n\nIt should be discussed about the software used, would the results be different if the clinician used the different software?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-166
|
https://f1000research.com/articles/8-173/v1
|
11 Feb 19
|
{
"type": "Research Note",
"title": "Comparative evaluation of sensitivity and specificity of immunochromatography kit for the rapid detection of norovirus and rotavirus in Bangladesh",
"authors": [
"Modhusudon Shaha",
"Sadia Farzana Sifat",
"Md. Al Mamun",
"Md. Baki Billah",
"Nadim Sharif",
"Nasir Uddin Nobel",
"Anowar Khasru Parvez",
"Ali Azam Talukder",
"Akiko Nomura",
"Hiroshi Ushijima",
"Shuvra Kanti Dey",
"Modhusudon Shaha",
"Sadia Farzana Sifat",
"Md. Al Mamun",
"Md. Baki Billah",
"Nadim Sharif",
"Nasir Uddin Nobel",
"Anowar Khasru Parvez",
"Ali Azam Talukder",
"Akiko Nomura",
"Hiroshi Ushijima"
],
"abstract": "We report a comprehensive analysis of sensitivity and specificity of immunochromatography kit (IC Kit) for the rapid detection of norovirus and rotavirus in Bangladesh. The IC kit (IP-Noro/Rota) provides highest sensitivity (100%) to both viruses compared to the reference method reverse transcription- polymerase chain reaction (RT- PCR) for diagnosis. Furthermore, the test provides a high specificity of 98.9% and 96.1% to diagnose norovirus and rotavirus, respectively, as well as good agreement with the reference method. We also found high prevalence of rotavirus infection (74%) among Bangladeshi pediatric population, of which most of the patients were less than five years old, suffering from severe dehydration, abdominal pain and vomiting. This study is the first to report the ease and rapid detection of norovirus and rotavirus by IC kits in Bangladesh. Therefore, IP-Noro/Rota kit is recommended for the rapid detection of these viruses in routine diagnosis as well as during outbreaks.",
"keywords": [
"Immunochromatography kit",
"Norovirus",
"Rotavirus",
"Rapid detection",
"Bangladesh."
],
"content": "Introduction\n\nDiarrheal diseases represent a major worldwide public health problem, particularly in developing countries1. Acute gastroenteritis is a very common disease in young children2. It has been reported that about 3–5 billion cases of acute gastroenteritis occur each year in children less than 5 years old and 1.5 to 2.5million children of that group die from severe diarrhoea3–5. Of that, about half amillion death is caused by rotavirus infections6. On the other hand, norovirus is responsible for almost half of the foodborne gastroenteritis outbreaks and 75–90% of non-bacterial gastroenteritis outbreaks7.\n\nWhen outbreaks of gastroenteritis occur in communities, rapid identification of pathogens is essential to ensure the administration of the appropriate treatment and control. Furthermore, definite diagnosis plays an important role to decrease the unnecessary use of antibiotics. In the case of emergency, there is no rapid detection method available in Bangladesh. In this regard, a rapid diagnosis kit with good sensitivity and specificity is essential. Developing such a kit may raise the reliability for rapid diagnosis in developing countries, where the prevalence of norovirus and rotavirus is increased. Herein, we report a comprehensive analysis of the sensitivity and specificity of an immunochromatography kit (IC Kit) for the rapid detection of norovirus and rotavirus in Bangladesh.\n\n\nMethods\n\nIn this study, we evaluate the newly developed IC test kit for norovirus and rotavirus detection (IP-Noro/Rota; ImmunoProbe Co., Ltd., Saitama, Japan) in 100 stool samples collected from pediatric patients with acute gastroenteritis (severe dehydration, abdominal pain and vomiting) in Bangladesh during January to June 2015. The study was ethically approved by the ethical review committee of Jahangirnagar University, Bangladesh.\n\nReverse transcription PCR (RT-PCR) was used as the reference test for both norovirus and rotavirus detection3. The PCR is a molecular biology technique that allows for nucleic acid fragment from a complex pool of DNA. Faecal specimens were thawed, diluted with distilled water to 10% suspensions, and centrifuged at 10,000xg for 10 min. Viral RNA was extracted from 140µl of the supernatant using a spin-column technique (QIAamp Viral RNA kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For reverse transcription, 3µl of extracted RNA was mixed with a reaction mixture consisting of 1µl of oligo dT primer (Promega, Madison, USA) and 1µl of nuclease free water in microcentrifuge tube, then kept at 70°c for 5 mins and then chill for 5 mins. After that 4µl of 5X reaction buffer (Promega, Madison, USA), 2µl of MgCl2, 1µl of PCR Nucleotide Mix (Promega, Madison, USA), 0.5µl of Ribonuclease Inhibitor (Promega, Madison, USA), 1µl of Reverse Transcriptase (Promega, Madison, USA), 6.5µl of nuclease free water were mixed with the same microcentrifuge tube. Then the solution was heated at 25°C for 5 mins, 42°C for 60 min and 70°C for 15 mins. Norovirus and rotavirus were detected by PCR analysis of cDNA with specific primers previously published8–9. The amplification was carried out in a thermal cycler (2720 Thermal Cycler, Applied Biosystems, USA). The PCR was performed at 94°C for 3 mins followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 60 s and a final extension at 72°C and then held at 4°C. The PCR products were electrophoresed in a 1.5% agarose gel, followed by staining with ethidium bromide (0.5 g/ml) for 20 min and then visualized under ultraviolet (UV) light. The bands were recorded by photography.\n\nTo evaluate the sensitivity and specificity of this IP-Noro/Rota test kits, all the 100 samples were tested for norovirus and rotavirus antigens by this kit following manufacturer’s instructions (IP-Noro/Rota; ImmunoProbe Co., Ltd., Saitama, Japan). It took only 10-15 min to obtain the result. A positive result for both pathogens is two lines; the left control line (C) and the right test-positive line (T), whereas, a negative result consisted of a single left control line (C) (Figure 1).\n\nThe test is positive if two lines appear in the membrane (a). The test is negative when only one line appears in the control area (b).\n\nThe sensitivity and specificity of IP-Rota/Noro test kit were calculated mathematically as described below:\n\nSensitivity for IC kit = Both IC kit and RT-PCR Positive × 100/ RT-PCR positive\n\nSpecificity for IC kit = Both IC kit and RT-PCR negative × 100 / RT-PCR negative\n\n\nResults\n\nThe working plan for evaluation of sensitivity and specificity of immunochromatography methods for rapid detection of rotavirus and norovirus associated with paediatric diarrhoea in Bangladesh is described in Figure 2.\n\nBy the RT-PCR method, 10 and 74 samples were confirmed as norovirus and rotavirus, respectively. It was found that all the isolated norovirus belongs to the genogroup II (data not shown). On the other hand, G1P8 rotavirus strain was found the most prevalent among the Bangladeshi pediatric population after characterization of G-types (VP-7) and P-types (VP-4) of rotavirus-positive samples. The youngest patient was 21 days and the oldest 56 months; the average age was 14 months. The most common clinical symptoms of rotavirus and norovirus infected patients were dehydration, vomiting, fever and abdominal pain.\n\nOf the 10 and 74 samples positive for norovirus and rotavirus, respectively, by RT-PCR, IP- Noro/Rota kit recognized all positive samples with 100% sensitivity. However, the kit gave one false positives for norovirus and three false positives for rotavirus detection, resulting in a specificity of 98.9% and 96.1%, respectively (Table 1 and Table 2).\n\n\nConclusions\n\nThe clinical symptoms of the patients with acute gastroenteritis are generally not indicative of a specific pathogen. In Bangladesh, the outbreak of norovirus and rotavirus diarrhea occurs mainly in the winter season3, when the IC kits could be used for rapid screening, as other existing diagnosis methods are time consuming. The rapid IC kit test is easy to perform at a low cost and it takes only 10–15 min to diagnose with a simple procedure and does not require special equipment or a skilled technician.\n\nOur findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only. The IC kit provides a high specificity and sensitivity as well as good agreement with the reference method, RT-PCR, for the detection of rotavirus and norovirus. Therefore, IC-Noro/Rota kit will be easy and useful assay for the rapid detection of these viruses in routine diagnosis as well as during the outbreaks. This is the first report about the rapid detection of rotavirus by IC kits in Bangladesh. Finally, it is strongly recommended to use the IC kit as an alternative method for rapid diagnosis of norovirus and rotavirus infections, especially in developing countries like Bangladesh.\n\n\nData availability\n\nFigshare: Raw Data- IC kit.csv, https://doi.org/10.6084/m9.figshare.7616630.v110.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThis research was supported by Grants-in-Aid from the Ministry of Education, The People’s Republic of Bangladesh.\n\n\nAcknowledgements\n\nWe thank the ImmunoProbe Co., Ltd. (Saitama, Japan) for kindly providing the IP-Rota/Noro kit.\n\n\nReferences\n\nCheng AC, McDonald JR, Thielman NM: Infectious diarrhea in developed and developing countries. J Clin Gastroenterol. 2005; 39(9): 757–773. PubMed Abstract | Publisher Full Text\n\nHowidi M, Al Kaabi N, El Khoury AC, et al.: Burden of acute gastroenteritis among children younger than 5 years of age--a survey among parents in the United Arab Emirates. BMC Pediatr. 2012; 12: 74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDey SK, Nguyen TA, Phan TG, et al.: Molecular and epidemiological trend of norovirus associated gastroenteritis in Dhaka City, Bangladesh. J Clin Virol. 2007; 40(3): 218–223. PubMed Abstract | Publisher Full Text\n\nDey SK, Phan TG, Mizuguchi M, et al.: Genetic diversity and emergence of norovirus GII/4-2006b in Japan during 2006-2007. Clin Lab. 2011; 57(3–4): 193–199. PubMed Abstract\n\nChow CM, Leung AK, Hon KL: Acute gastroenteritis: from guidelines to real life. Clin Exp Gastroenterol. 2010; 3: 97–112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTagbo BN, Mwenda JM, Armah G, et al.: Epidemiology of rotavirus diarrhea among children younger than 5 years in Enugu, South East, Nigeria. Pediatr Infect Dis J. 2014; 33 Suppl 1: S19–22. PubMed Abstract | Publisher Full Text\n\nBarreira DMPG, Fumian TM, Tonini MAL, et al.: Detection and molecular characterization of the novel recombinant norovirus GII.P16-GII.4 Sydney in southeastern Brazil in 2016. PLoS One. 2017; 12(12): e0189504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYan H, Yagyu F, Okitsu S, et al.: Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR. J Virol Methods. 2003; 114(1): 37–44. PubMed Abstract | Publisher Full Text\n\nDey SK, Thongprachum A, Ota Y, et al.: Molecular and epidemiological trend of rotavirus infection among infants and children in Japan. Infect Genet Evol. 2009; 9(5): 955–61. PubMed Abstract | Publisher Full Text\n\nShaha M, Sifat SF, Al Mamun Md., et al.: Raw Data- IC kit.csv. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.7616630.v1"
}
|
[
{
"id": "44319",
"date": "20 Feb 2019",
"name": "Mustafizur Rahman",
"expertise": [
"Reviewer Expertise Virology",
"Infectious diseases",
"Rotavirus vaccine",
"norovirus epidemiology",
"viral hepatitis",
"HIV"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluate a newly developed immunochromatography (IC) kit for detecting norovirus and rotavirus by comparing the results with Reverse transcription PCR method as gold standard. They conducted comprehensive analysis of the sensitivity and specificity of the IC kit and concluded that the kit could be performed for rapid detection of norovirus and rotavirus with low cost and recommended the kits for routine use in low-income countries like Bangladesh.\n\nMajor comments: 1. Lack of information about sample quality: Authors claimed that the samples were collected from January to June 2015 and stored in -20⁰C, but did not mention the duration of storage in the freezer before testing, which could affect the evaluation. The reviewer suggests to use freshly collected samples for this type of comprehensive analysis. 2. Selection of inappropriate gold-standard for comparison: Authors selected Reverse transcription PCR (RT-PCR) for comparison. This PCR depends on primers which were designed more than 15 years ago (published in 2003); however, according to recent data and considering high diversity of the viruses, these primers may fail to detect the viruses correctly in many samples. Thus, this assay cannot be used as gold standard. Using more reliable and latest detection methods such as real time RT-PCR can be used as a gold standard. 3. Miscalculation: Analysis for sensitivity and specificity was incorrectly performed. For example, specificity of the kit for rotavirus detection (in Table 2) should be 89.6%. 4. Concluding remarks: The statement “Our findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only.” cannot be made from the data presented in this paper. This study was particularly designed to evaluate a kit; not for describing rotavirus and norovirus prevalence. The authors strongly recommended to use the IC kit as an alternative method for rapid and low-cost diagnosis without the cost analysis by comparing with other available detection kits. Mentioning the unit cost for this IC test could be helpful to support this statement.\n\nOne of the major limitations of this study is the lack of information on the capability of the IC kit to detect different genotypes of viruses by this IC kit. All norovirus strains detected in this study belonged to norovirus genogroup II and other norovirus genogroups were ignored. For example, in Bangladesh, about 15-30% of noroviruses belong to genogroups other than genogroup II1,2. Similarly, the author did not mention genetic variation of rotaviruses detected in this study except G1P3. A well-designed study by including different genotypes with appropriate sample size is recommended for this type of kit evaluation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5259",
"date": "02 Mar 2020",
"name": "Modhusudon Shaha",
"role": "Author Response",
"response": "I would like to thank the respectable reviewer for his valuable comments. The point-by-point response is given below- The laboratory analysis was conducted at the time of collection of the samples. After analysis of the samples, the leftover is stored for long time and is still available in the lab. Before selecting the primer pairs, a quality check was performed using the existing Genbank sequences of respective viruses. The primer oligo location in the viral genomes seems to be conserved. The corrected calculations are given in the revised manuscript. The statement is updated in the revised manuscript. The cost analysis was not performed, but the price of the kit would be much lower compared to the current diagnostic cost. The kit is developed to detect all genotypes of underlying viruses, not a specific genotype. This is a short research note to report the sensitivity and specificity of the kit against rotavirus and norovirus. However, reviewer’s suggestion will be considered in the next study."
}
]
},
{
"id": "45947",
"date": "26 Mar 2019",
"name": "Nicola A. Page",
"expertise": [
"Reviewer Expertise Viruses associated with diarrhoeal diseases",
"rotavirus vaccines",
"molecular characterization and phylogenetics of enteric viruses",
"epidemiology and public health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper reports the performance of an immunochromatography cartridge for the rapid detection of norovirus and rotavirus against an RT-PCR reference method. The specimens selected (n=100) were collected between January and June 2015 in Bangladesh and had been frozen prior to use. The authors also calculated the sensitivity and specificity of the kit and made recommendations regarding the potential use. However, the paper has a few problems:\n1. The calculations in Table 1 and 2 are incorrect. The sum of the rows and columns do not total 100. Consider correcting the tables an indicated below.\n\nTable 1. Comparison of norovirus detection in stool samples by IP-Noro/Rota kit and RT-PCR method (n=100) Test result\n\nRT-PCR IP-Noro/Rota kit\n\nPositive\n\nNegative\n\nTotal Positive\n\n10\n\n1\n\n11 Negative\n\n0\n\n89\n\n89 Total\n\n10\n\n90\n\n100\n\nTable 2. Comparison of rotavirus detection in stool samples by IP-Noro/Rota kit and RT-PCR method (n=100) Test result\n\nRT-PCR IP-Noro/Rota kit\n\nPositive\n\nNegative\n\nTotal Positive\n\n74\n\n3\n\n77 Negative\n\n0\n\n23\n\n23 Total\n\n74\n\n26\n\n100\n\nThe calculations for specificity are, therefore, incorrect. This should be correct in the next version of the paper.\n2. The authors briefly mention the broad typing results for norovirus (genogroup II) and rotavirus (G1P[8]) but fail to give any indication of the methods used to obtain these results. It makes it difficult for the reviewers and readers to assess the results presented. In addition, no detailed genotyping results were presented for the viral strains detected. Most current papers evaluating diagnostic assays for enteric viruses include genotyping data for the virus strains detected - see references below.\n\n3. The authors failed to report the manufacturers’ sensitivity and specificity for the kit or to compare their results to other studies that evaluated the same or similar kits – see articles below. Alternatively, if this is a new formulation of the kit being evaluated the authors should indicate this fact.\n4. The authors utilized stool specimens that were frozen for an unspecified period (but approximately three years if the study was completed in 2018). The prolonged storage or repeated freeze/thawing could have contributed to a degree of degradation of the viruses in the stool material and should have been listed as a potential limitation of the study. However, the screening of the frozen stool specimens is a previously utilized approach for evaluating diagnostic kits.\n5. If the kit is introduced according to the author’s recommendations, it is more likely that fresh stool specimens will be screened. Therefore, the authors should consider evaluating the kit using fresh stool specimens from routine surveillance or during an outbreak which would mimic real world use and provide better estimates of sensitivity and specificity.\n\n6. Based on the detection rate of rotavirus in the stool specimens selected for the evaluation (74%), these specimens were collected during the rotavirus season in Bangladesh. While the results indicate that the kit would be adequate for use during the rotavirus season (barring a few false positive results), it does not give any indication of the kits performance when the rotavirus prevalence declines, outside of the rotavirus season.\nAdditional comments:\nThe term “developing country” is imprecise and inaccurate with no standard definition of what constitutes a developing country. The World Bank has defined standards for classifying countries so a better definition or term is a low-income country. Please correct in paragraphs 1 and 2 of the Introduction and in the last paragraph of the conclusions.\n\nPlease check your nomenclature for rotavirus genotypes – G1P[8] and not G1P8. The statement “Our findings clearly indicate that rotavirus and norovirus are the most important enteropathogen responsible for acute viral gastroenteritis among infants and children in Bangladesh, where 74% of the cases were caused by rotavirus only” is not valid. The authors only screened for rotavirus and norovirus. Other enteric viruses may also be important in gastroenteritis in children in Bangladesh but because the authors failed to screen for these pathogens, these observations would have been missed and, therefore, conclusions regarding viral enteropathogen importance are incorrect.\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? No\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": "5260",
"date": "02 Mar 2020",
"name": "Modhusudon Shaha",
"role": "Author Response",
"response": "I would like to thank the respectable reviewer for the constructive comments and valuable suggestions. The revised manuscript is corrected based on the reviewer’s comments. The point-by-point response is given below- The corrected tables are given in page 5 and 6 of this revised manuscript. This is a short research note. The genotyping was obtained by sequencing and the phylogenetic tree is not shown here in this manuscript. The results of this study are compared with the existing reports and the results part is updated in page 5. The laboratory analysis was conducted at the time of collection of the samples. After analysis of the samples, the leftover is stored for long time and is still available in the lab. The evaluation of the kit was tested using the fresh samples. The kit gave a specificity of 100% as compared to the RT-PCR analysis, though some false positive results may have been given. The statement is updated in the revised manuscript. Additional corrections- -In all the paragraphs, the term “developing countries” is replaced by “low-income countries” in the revised manuscript. -The nomenclature of the rotavirus genotypes is corrected. -The statement is updated in the revised manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/8-173
|
https://f1000research.com/articles/8-1854/v1
|
05 Nov 19
|
{
"type": "Opinion Article",
"title": "The why, when, and how of computing in biology classrooms",
"authors": [
"April M. Wright",
"Rachel S. Schwartz",
"Jamie R. Oaks",
"Catherine E. Newman",
"Sarah P. Flanagan",
"Rachel S. Schwartz",
"Jamie R. Oaks",
"Catherine E. Newman",
"Sarah P. Flanagan"
],
"abstract": "Many biologists are interested in teaching computing skills or using computing in the classroom, despite not being formally trained in these skills themselves. Thus biologists may find themselves researching how to teach these skills, and therefore many individuals are individually attempting to discover resources and methods to do so. Recent years have seen an expansion of new technologies to assist in delivering course content interactively. Educational research provides insights into how learners absorb and process information during interactive learning. In this review, we discuss the value of teaching foundational computing skills to biologists, and strategies and tools to do so. Additionally, we review the literature on teaching practices to support the development of these skills. We pay special attention to meeting the needs of diverse learners, and consider how different ways of delivering course content can be leveraged to provide a more inclusive classroom experience. Our goal is to enable biologists to teach computational skills and use computing in the classroom successfully.",
"keywords": [
"computation",
"biology",
"education",
"undergraduate"
],
"content": "Introduction\n\nBiology departments are increasingly offering or considering course work that incorporates hands-on computation (Wilson Sayres et al., 2018). There are different rationales for courses that include computation. Perhaps the most obvious rationale is to teach computational biology, bioinformatics, and/or skills in working with large datasets. Bioinformatic skills are increasingly a core component of biology and biology education, and support students’ job and/or graduate school preparation (Wilson Sayres et al., 2018). The vast majority of researchers in biology either use large datasets or could do so with sufficient training, suggesting that general training in effective use of large datasets is essential for students as well (Barone et al., 2017; Loman & Watson, 2013). Thus, in such courses, instructors teach particular skills or tools that are an integral part of the field. An alternate motivation is to incorporate more data and analysis into course work. Integrating data into courses provides more opportunity for students to experience the research process, and the ability to incorporate active learning activities where students recognize biological theories by drawing conclusions from their own results (Kjelvik & Schultheis, 2019). Another rationale for teaching computational skills is to increase data literacy (Gibson & Mourad, 2018), which, in addition to enhancing learners’ preparation for future careers, also better prepares learners to think critically about societal issues relevant to science and policy (Cook et al., 2014).\n\nHowever, there are significant barriers to teaching hands-on computation in biology classrooms (Williams et al., 2017). While many biologists use computation in their research, relatively few have explicit training in this area. Fewer still have training in how to teach computation (Williams et al., 2017). Importantly, few biologists are housed in a department with the existing expertise or infrastructure for teaching computation (Williams et al., 2017); thus, there is often less familiarity with options for teaching computing or recent best practices. Cummings & Temple (2010) describe the challenges in teaching bioinformatics as “infrastructure and logistics; instructor knowledge of bioinformatics and continuing education; and the breadth of bioinformatics, and the diversity of students and educational objectives.” Williams et al. (2017) similarly cites faculty training, infrastructure, and student interest and preparation. Here our goal is to address issues of infrastructure and logistics, and make recommendations for faculty practice. We suggest that those seeking training consider programs such as Software and Data Carpentry workshops to address the issue of faculty training. This article explains options available for instructors to teach computational course content (collectively referred to here as computing platforms), discusses how to choose the best computing platform for a course, and describes effective strategies for teaching coding and data analysis.\n\nAn important consideration is that whichever technology an educator uses to deliver their content supports the educator’s learning objectives and enhances students’ ability to learn. Every course has a unique set of learning objectives, and every group of learners poses a unique set of challenges. Such learning objectives related to computing may range from engaging students in the value of basic coding skills to building advanced skills and the ability to self-teach and work independently. An example of the former might include a freshman introductory biology lab focusing on data analysis, where students focus on the results from the data analysis. In contrast, an upper-division course on research methods may focus more on preparing students for independent data analysis by emphasizing self-sufficiency in coding, and software management and installation. Additionally, the selected platform needs to support the type of work the students are doing. In the first example, students might need to learn to make appropriate plots to visualize biological data; thus, the platform students work with needs to have the capacity to display plots. On the other hand, if a course objective is for students to learn to manage genome-scale data on a remote server, a platform using a command-line interface to access a computing cluster may be more appropriate.\n\nMuch as a biology laboratory course requires specific laboratory equipment, teaching computation requires learners to be able to access specific software and functions. This may range from one or a few pieces of software that can be downloaded from the internet to installation of a computing language and development environment. For simplicity, the interfaces used by learners and the instructor should be uniform, as different versions of software could have different interfaces or commands. Learners also need a way to compute outside of class — for example, to do the homework outside class hours. Finally, the computing platform should allow students to focus on the coursework and instruction. The learning platform must not interfere with students’ focus on the learning objectives.\n\nAnother consideration is the consistency and scaffolding in selecting tools and/or the larger platform. Gibson & Mourad (2018) cites the need for “coordinated, sequential development of data literacy across the biology curriculum”, which suggests the value of a platform and tools on which students can scaffold their skills, rather than each course choosing its own tool. For example, students would likely find it challenging to use R in one class, Python in another (Wu & Anderson, 1990), and a genomics platform such as Galaxy in another. Furthermore, switching languages and platforms (i.e. learning a new skill set) can detract from time spent on other educational activities when the goal is not to explicitly teach the language or platform.\n\nEvery choice of computing platform comes with costs and benefits. In this article, we will discuss tools and techniques to facilitate teaching computation in courses with some degree of programming or data analysis. We explain different computing platforms with a special eye towards serving diverse sets of learners, in terms of their motivations, life experiences, and access to technology. In particular, we will focus on programming languages that are common in biology: R and Python. Lastly, we review effective pedagogical techniques for helping learners thrive in a computational classroom.\n\n\nChoice of computing language\n\nOne of the first choices an instructor needs to make when they plan coursework is what computing language to use. In biology, R and Python are common languages. Bash, a type of UNIX command-line language, is commonly used to run software and automate tasks. There are multiple considerations to take into account for this choice, including consistency, current status of research tools, and comfort. Consistency refers to consistency of the language’s use throughout the curriculum. Switching languages can be associated with learning loss in novices (Wu & Anderson, 1990). Therefore, in order to deepen and strengthen learners’ command of languages, integrating the same language consistently throughout the curriculum may be helpful. For example, if an instructor is integrating a computational component into a genetics lab, they might check with a colleague teaching biostatistics or bioinformatics to see what language they are using.\n\nThe current status of research tools is also worth considering. If the goal of a course is for learners to understand both biology and computation, the instructor will want to choose a language with active development of tools for research in that field. For example, many genomics and phylogenetics tools are written in Python. On the other hand, more ecological and comparative biology tools are written in R. Therefore, the choice of language will inform the subset of tools available to the instructor and the learners.\n\nFinally, instructors should consider their own comfort. Teaching requires fluent and comfortable discussion of concepts and tools. Instructor knowledge must factor in to this. Additionally, community resources exist for some languages. For example, QUBES is a National Science Foundation-sponsored (NSF) project that publishes lessons and sponsors instructor mentoring groups in various scientific disciplines. An instructor may feel more empowered to teach well if there is a strong sense of community in their language, with pathways to ask for help and access resources.\n\nIt is also worth noting that not all tasks that are computational necessitate the addition of programming into the curriculum. For example, tools such as the NSF-sponsored Galaxy project allow instructors to help learners run bioinformatics and genomics software via a point-and-click internet interface. If the goal of a course is to demonstrate answering a particular question, but not to dig too deep into the methodology of how that is done, this type of interface may be sufficient. Teaching with these resources is beyond the scope of this article, as the Galaxy website provides extensive resources for teaching, and trainers provide regular workshops on this tool.\n\nThe R language allows inexperienced learners to immediately work with rectangular (i.e. spreadsheet-style) data in a way that is engaging. For example, a learner can load data and plot results within an hour of first opening RStudio. This engagement is important for learners who are more interested in effectively working with data than learning to code. We suggest that the R language immediately supports coding-to-learn with minimal learning-to-code. In particular, the tidyverse set of packages provided by RStudio provides convenient, well-supported, and intuitive functions for plotting and manipulating data. Bioconductor (Huber et al., 2015) has a wide variety of packages related to genomic data, as well as tutorials for this use, while other packages such as ape (Paradis & Schliep, 2018) provide resources for specific tasks such as reading and viewing phylogenies, and many other packages are available with easy-to-follow vignettes.\n\nPython is a general purpose programming language used in a wide variety of industries and can be adapted to many purposes. For example, the popular data science library Pandas (McKinney, 2010) can be used for teaching foundational data skills. Python also has a variety of resources for teaching work with non-spreadsheet data. BioPython is a library for working with general sequence datasets at a variety of scales (Cock et al., 2009). More specialized libraries, such as Poretools (Loman & Quinlan, 2014) (for Nanopore data) or Dendropy (Sukumaran & Holder, 2010) and ETE3 (Huerta-Cepas et al., 2016) (for phylogenetic trees), are also available for work in subdisciplines of biology and bioinformatics.\n\nDepending on the aims of the class, it may be necessary to choose a supporting language, as well. For example, in contrast to a coding-to-learn course in which biology students practice drawing conclusions from data, a course on genomics might require students to interact with large datasets on a remote server. Many such servers require a UNIX command line to access. While this article will focus on R and Python, some instructors may find that they need additional languages to support student learning, particularly in advanced disciplines. Furthermore, while we suggest focusing on a single language for pedagogical reasons, and highlight R and Python, our goals in choosing a language are (1) to select one that is widely used with available tools, and (2) has straightforward syntax for novices (Stefik & Siebert, 2013). We support the philosophy taken by the Software Carpentry organization of using any language that meets these goals and provides a platform for learning the fundamental process of automating data analysis.\n\n\nLocal computing platforms\n\nPerhaps the most basic computing platform setup is to have learners install languages and software locally on their own computers. Local installs may work best in small classes that focus on the basics of coding, where the goal is for learners with no prior experience to leave the course self-sufficient to do computational work. R is easily downloaded and installed from the R-Project website. RStudio provides a commonly-used integrated development environment (IDE) that allows management of additional packages and visualization of data and figures. The Anaconda installation of Python provides the programming language, as well as the Jupyter notebook environment for simultaneously viewing code and output, and the ability to run code in blocks. The Anaconda installation also provides the conda package manager for installation of additional packages.\n\nAdvantages of local installs. Local installs allow learners to immediately be able to apply their new knowledge of code outside the class without additional support. For learners who are already familiar with their own computers and operating systems, having students use local installs can bypass some of the learning curve associated with learning a completely different platform; this also applies for instructors, who often have limited time to devote to learning a new platform exclusively for the purpose of teaching a course.\n\nIf the software used in class does not require a paid license or subscription, learners will continue to have access to the specific tools used in class after the course ends. Teaching learners with tools that they know they will be able to take away with them and apply in the future increases learner motivation to learn these tools, and increases the chance that they will actually use them in future work (Kember et al., 2008).\n\nLocal installs are a solution in particular for smaller institutions that do not have access to remote computing platforms. In some cases, though setting up a computing platform may be theoretically possible, it may not be feasible within the constraints of resources, time, and IT personnel. Local installs offer an alternative system that does not typically require extensive coordination with IT personnel at an institution.\n\nChallenges of local installs and how to overcome them. While using local installs can minimize time spent learning new platforms, it may also extend the initial time spent setting up learners’ computers for the course – namely, with language and software installation. The feasibility of using local installs may thus be limited in many cases by class size. This up-front time can be minimized by having learners complete as much installation as possible before the course begins using detailed instructions or videos available on the software developers’ websites or provided by the instructor. For example, Data Carpentry provides installation instructions as part of its workshops and the University of British Columbia provides installation information to support its Data Science courses. However, some guidance may be necessary either during class or individually. In addition, troubleshooting installs on various platforms can be challenging, especially with a larger class working on a wider variety of computers. In some cases, software may not be available for a particular platform (e.g. RStudio on Chromebooks). Further, if learners have previously installed a particular language, IDE, or package, they may have a different version than that used in class, which may complicate analyses later. This issue would present less of a problem in classes that primarily cover the foundations of coding, and/or with learners who have little to no prior experience.\n\nIn classes that have learners do analytical work requiring substantial computational power, learners may not have computers that are able to do that work. Some learners may not have computers at all. For these reasons, local installs may not be the best option for a required course (i.e., if a learner must take the course regardless of whether or not they have a computer they can bring to class) or for a course where a substantial amount of time is spent on computationally intensive work. For elective courses, the instructor may choose to list a computer as required material for the course and make that information available to students during the class registration period. However, this requirement limits the learners who are able to take the class to those who have a computer to bring. With small classes, it may be possible to provide a computer to a learner who does not have their own, and the instructor should also ensure that the learner has access to a computer outside the classroom with necessary software for homework. An alternative solution may be to hold the class in a computer lab, but installation of software in computer labs may require IT support, depending on the institution.\n\nSingle-board computers (SBCs) are minimalist, complete computers built on a single circuit board. There are a number of organizations in the single-board computer market, but they were first popularized by the Raspberry Pi Foundation. The most basic SBC models cost as little as $10 and offer WiFi, Bluetooth, and quite high-performance computing. Generally, a Linux distribution, such as Raspbian, is installed on a microSD card and run on these machines.\n\nAn SBC is not immediately useful on its own, because it does not have a monitor or input devices. However, these machines can be used (1) as a server, by logging in “remotely” from another machine via WiFi or wired connection — for example, using secure shell (SSH) protocol — or (2) as a standard Linux PC, by booting into the desktop environment and using attached input and output devices (e.g., monitor, keyboard, and mouse). Having learners use SBCs shares some similarities to using local installs, in the sense that the learners can run software installed on their own local machine. However, SBCs would ensure that all learners are using the same computing environment and have the same capabilities. Furthermore, SBCs can be used together with all the other computing platforms we discuss here.\n\nAdvantages of SBCs. Teaching with SBCs can be particularly effective at an institution with limited IT support. Even in a classroom without reliable internet, the instructor can set up an SBC as a local network for sharing code and data among the learners. Thus, they can also be used in teaching environments not traditionally thought of as computing-friendly, such as in the field.\n\nSBCs can be particularly good tools for teaching learners how to use remote Linux servers and HPC resources. Remote computing can be an abstract concept for students with limited computational background. Showing students how to “remotely” login from another computer to their SBC they have in-hand can make the idea of remote computing much more tangible. Furthermore, SBCs offer a way of teaching high-performance computing when such resources are not available. Even institutions with HPC facilities often do not support the use of these resources for teaching. On a very modest budget, an instructor can set up a shared, “mini” computing cluster to teach fundamental principles of HPC, such parallelization, message passing, and scalability.\n\nOne particularly exciting pedagogical opportunity presented by SBCs is the integration of tech and computation to create biology curricula needed by students in the 21st century (see Box 1). Imagine a scenario where all learners entering a program would receive an SBC along with some basic training on how to use it. Because SBCs allow direct access to the computing board and associated headers and ports, they are extremely modular, and can be coupled with many inexpensive accessories for collecting data. For example, for as little as $40, one can purchase an add-on board that includes a variety of sensors, including a gyroscope, accelerometer, magnetometer, temperature, barometric pressure, and humidity. Learners using these devices could collect observational or experimental data in the lab of their introductory biology classroom. Then, in a lecture or discussion session, they could learn to use basic scripting to process, visualize, “clean,” and analyze those data. If an undergraduate or graduate curriculum was designed for the students to continue using their SBC, data, and scripts throughout their coursework, this could be a powerful approach to training the next-generation of computationally competent biologists. The development of such a curriculum goes far beyond the scope of what a typical teacher needs from a computing platform. Nonetheless, this aspirational idea is worth highlighting when discussing SBCs, because they can be used jointly with all the other computing platforms discussed in this paper.\n\nThe low cost of high-performance, single-board computers (SBCs) can make the classroom more equitable. With a cost as low as $10 per computer, it requires minimal investment from learners, or, more ideally, increases the likelihood that an institution or funding agency can cover this cost. Using SBCs also ensures that all learners experience the same computing environment and capabilities. Furthermore, the low cost can allow the learners to keep the computer after the course is over. This can enable life-long learners across all socioeconomic backgrounds.\n\nDisadvantages of SBCs. The initial setup of an SBC requires time and effort to install the Linux distribution on the microSD card. If the primary goal of the course is to use technology to teach biology, this work should be done by the instructor prior to the class. If the class is large, this would most likely require IT support to pre-install the operating system on many microSD cards. However, if any of the learning objectives of the course involve a better understanding of operating systems or computer hardware, turning the initial setup into an active-learning exercise done at the beginning of the class could be a very informative learning experience for the students.\n\nEven after the initial setup, SBCs are not immediately useful on their own. Learners require either another computer to login to the SBC via SSH, or peripherals (e.g., monitor, keyboard, and mouse) to use the SBC as a PC. By the time these peripherals are factored in, the total cost could become similar to an entry level laptop. However, the learners can benefit from the modularity of the SBC system. The peripherals can be re-used, and the students could keep the SBC itself. This is not possible with laptops.\n\nWhen not used as a PC (i.e., without peripherals), using an SBC can be quite unintuitive for learners not familiar with the Linux command line. For example, it would be counter-productive to take the time necessary out of an introductory biology class to teach learners to become proficient in using an SBC from the command line. While this weakness can be overcome by using the SBC as a PC, this option does require additional investment and logistics to provide the learners access to keyboards, mice, and monitor.\n\n\nCloud-based computing platforms\n\nA number of platforms for serving course materials via the internet have become available. Because these technologies share many similarities, we will first discuss these computing platforms generally, then discuss teaching with R (such as with RStudio server, per RStudio Team (2015)) and Python (such as with JupyterHub, per Kluyver et al. (2016)) in the cloud.\n\nCloud-based systems typically provide a web interface to a centralized version of an IDE. This allows the instructor to set up a uniform instance for all learners, avoiding time spent sorting out problems, such as learners having different versions of languages or software installed. Learners can then focus on learning biology and/or working with data, rather than on managing technical details.\n\nCloud-based computing platforms also ensure that learners are not limited by their hardware or software. The instructor sets up the class software on a server, rather than the learners installing software on their own personal machines, thereby avoiding problems such as learners not having computers powerful enough to run exercises, or computers without the ability to do language installs (such as NetBooks). This can be especially important for serving students who have difficulty purchasing a personal laptop to bring to class (see Box 2).\n\nA local RStudio Server or JupyterHub reduces barriers to the use of R or Python and support underprivileged students by removing the requirement to have a laptop (a Chromebook or tablet is sufficient) and minimizes the frustration associated with installation and different operating systems. Students can access the server from any location via a web browser using any machine while experiencing a consistent interface and access to their code and data.\n\nMany cloud-based computing platforms are agnostic to the type of machine on which they are run. For example, JupyterHub and RStudio Server can be deployed on any machine with Unix or Linux supported (RStudio Team, 2019). This machine can be owned by an instructor, a high-performance cluster (HPC) computer operated by a university, or cloud computing resources rented by the instructor. The main considerations for choosing what type of machine to run a cloud-based computing platform are memory, storage, and access. For courses with low-memory tasks, such as data processing on small datasets, a single machine may be sufficient, even with large numbers of users. For high-memory tasks (such as genome assembly), or tasks that with long runtimes (such as phylogenetic estimation), cloud compute resources that can be resized from week to week may be more suitable. For example, platforms such as Digital Ocean and Amazon Web Services allow users to pay for more memory only when needed. The server can be resized between class periods to allow more memory to be available to learners. This flexibility can allow an instructor to run a course on the resources they have available to them, based on their available infrastructure and funds.\n\nMany faculty will require IT support to set up the server, especially if the server is used by a large population. However, in our experience once setup is complete, maintenance requires minimal time and having IT handle upgrades and additional users relieves faculty of some work. The size of the server required for even large numbers of students to do analyses can be quite minimal (e.g. just 4 cores for hundreds of students), provided they are conducting analyses of relatively small datasets (e.g. hundreds of samples).\n\nWhile the cloud-based computing platforms are a flexible tool for teaching, there are some downsides. Because students typically interact with the server via a web-based login, if university resources are to be used, instructional technology (IT) staff will typically want to be involved. Some universities do not allow public-facing servers to be operated by non-IT staff, which can limit instructors from using computers they have on hand to serve their coursework. Likewise, due to security concerns, it may not be possible to operate a server on a university HPC. In this case, the instructor will likely have to turn to cloud compute providers. Many of these providers are affordable for small classes or low-memory tasks, but costs will scale with users, storage, and memory requirements.\n\nThese technologies do interact with the internet. Therefore, when problems arise in the classroom, the instructor must distinguish between problems with the server itself, with a learner’s computer, and with other classroom technology, such as wireless internet. While internet-based technologies can increase for equity and inclusion by allowing learners to interact with the course materials no matter how old their computer is, or if learners have unreliable computer access, they increase the complexity of diagnosing performance problems. Unstable internet connections can cause many, if not all, members of the class to lose connection to the materials during class. Learners with individual computer problems, such as malfunctioning wireless cards, may need to borrow a computer to access materials. If the instructor is teaching without IT support, they may need to halt class and fix problems if the server itself is malfunctioning.\n\nAdditionally, because a server facilitates learners focusing on the data and analyses, rather than installation and troubleshooting, learners may leave class without the ability to work independently outside the classroom. They have not learned how to install the programming language or IDE, and these are not installed on their personal computer. Additionally, they do not have or know how to install supporting packages. If the goal is for students to be equipped for independent work, the instructor should be sure to introduce these skills explicitly during the semester.\n\nRStudio Server (RStudio Team, 2019) provides a web interface to a centralized version of the RStudio IDE for working with R code. By using RStudio learners are able to simultaneously view their script, environment variables, file structure, plots, installed packages, etc. The Server format allows the administrator to maintain a consistent version of R, RStudio, and installed packages. This approach allows beginning coders to focus on data and results, rather than worrying about installation and versions. It allows an instructor to be sure that learners can focus on their code without being limited by their hardware or software.\n\nSome advantages of RStudio Server are that learners’ data and code are stored (and backed up) on the server. Instructors can also access a student’s code from anywhere to help students solve problems remotely. RStudio Server is free for teaching purposes upon submission of a syllabus to the RStudio Company. Additionally, for smaller scale teaching an instructor may use RStudio Cloud, which provides a free ready-to-go web-based version of RStudio.\n\nProject Jupyter is a non-profit organization that creates open-source tools for computation. One of their most famous products is the Jupyter Notebook, which allows researchers to create interactive code documents. These documents can have text, images, and code cells that run and render output for inspection. The Jupyter Notebook was originally developed as the iPython notebook, but has since expanded to include language support for other popular languages such as R and Julia. A JupyterHub is a server on which an instructor can deliver lessons via Jupyer Notebooks to a full classroom of students using a standardized server environment. In most cases, the server will be accessed via the internet. The first and foremost reason to use a JupyterHub is that you want to teach with the Jupyter ecosystem of tools. The Jupyter Notebook is commonly used in education in the sciences. It allows the instructor to develop a set of instructional materials that include text, code, and images. In this way, detailed notes can be provided from the instructor to the learner, and the learner can augment these lessons with their own notes, annotations, and code. Though originally invented for use with Python, Jupyter Notebooks are compatible with a number of languages. Multiple languages can even be used in one notebook, making them ideal for use with multi-language pipelines. The JupyterHub also has a command-line terminal and the Git version control system installed, for more advanced courses.\n\nJupyter supports the R language. However, the interface for R in Jupyter is different than RStudio, a dominant way to interact with R. Likewise, the JuptyerHub does not support other popular integrated development environments (IDEs), such as Python’s Spyder. While there is a text editor for preparing scripts, and a command line to run scripts, the JupyterHub is really designed to serve Jupyter Notebooks, and the interface caters to this technology.\n\nRStudio Server and JupyterHub will typically require that instances are hosted somewhere. This refers to having the compute platform installed in a central location, at which it can be accessed by all students. It may be possible for an instructor to host their course platform on a computer or server they personally own. However, in the experience of the authors, many universities prohibit faculty from hosting servers on-campus that will be available off-campus due to security concerns with allowing off-campus computers to access on-campus resources. This means that you may be able to host a server via the intranet, but if students go home for the weekend, or live off-campus, they’ll need to come back on to campus to do their homework.\n\nThese limitations cause many instructors to look for alternative ways to provide their course platforms to students. One common way is by working with a local high-performance cluster compute facility. A high-performance cluster computer is a set of computers that are networked. This allows researchers to harness the power of several computers at once. These facilities often also service educators. A benefit to serving course content via high-performance cluster computing is that these resources are often free for educators at the university where the computer resides. Because these services are often in-house, the administrators of them are able to handle any specialized security concerns with the course platform. National level high-performance cluster computers, such as CIPRES (Miller et al., 2010), JetStream (Stewart et al., 2015; Towns et al., 2014) and CyVerse (Goff et al., 2011; Merchant et al., 2016) also offer resources for US institutions. For campuses with limitations on HPC use, IT may be able to set up an independent virtual machine on other existing university servers.\n\nHowever, not every university has a high-performance cluster computer that is set up to host coursework. In this case, an instructor may want to consider a cloud-based solution. The concept of a cloud-based solution is similar to using high-performance cluster computing. An educator can rent one or more computers to serve their coursework. Cloud computing can take several forms. In some cases, the instructor explicitly rents computers in sizes that are appropriate for their course. These types of services often charge by the number of computers, the size of the hard drive rented, the amount of memory on the computer, and the amount of time for which they will run. Examples of this type of service include Digital Ocean, Cloud Flare, Google Cloud, and Amazon Web Services. However, it is important to notet that if you are signing a contract for cloud services, it is prudent to check with your IT, legal, and purchasing departments. Departments may have rules in place for student privacy and data security, as well as for contract bids between providers.\n\nRecently, educational cloud service providers have entered the market. Examples of these services include RStudio Cloud and Python Anywhere. These types of products are oriented towards providing an environment that is oriented towards a specific language or course platform. For example, RStudio Cloud hosts RStudio instances, and Python Anywhere hosts a variety of Python environments, including Jupyter notebooks. Like an regular cloud provider, these services typically charge based on the size of the amount of memory and storage capacity of the computer needed. Being oriented towards education, some of these providers also offer accounts for free, with paid accounts available for users who need more memory or computer power.\n\nContainer software, such as Docker, Singularity, or Podman, offer a way to teach with the simplicity of local installs while utilizing the consistency of cloud-based computing platforms. Containers allow the instructor to create a standard computing environment that each student can work in on their own computer. Instead of having to install multiple tools that will be used in the course, the students would only have to install the container software (e.g., Docker, which is cross-platform), and then they can download and run the environment created by the instructor. The functionality is very similar to a virtual machine, but lighter-weight and more efficient. Containers can be easily updated and distributed with cloud-based services like DockerHub. This allows the instructor to update the students’ computing environment in real-time. Of course, this still has the same disadvantages discussed above of having students run analyses on their own computer.\n\n\nTeaching methods\n\nDesigning an effective biology course that incorporates computing requires more than choosing the best computing platform for your needs. Skills-based courses such as coding necessitate a unique approach to teaching that can differ in some ways from more commonly used pedagogy in content-based courses. For many learners, a biology course that involves hands-on computing may be their first classroom experience that heavily involves learning increasingly complex skills that build off of each other, rather than primarily learning content. And similarly, instructors whose research and teaching expertise is in a field that is not strictly computational — which likely includes most instructors of biology — may benefit from guidance on best practices for delivering content effectively and addressing the unique challenges that arise. As a skills-based discipline, computation necessarily requires some level of active in-class interaction from learners (with the instructor or peers, or with the material itself). Thoughtful implementation of appropriate active learning strategies has been shown to increase both mastery of content and development of general skills such as critical thinking and collaboration (Faust & Paulson, 1998). Below, we describe several strategies for teaching coding, and address challenges that instructors may face in teaching a course that is perhaps more similar methodologically to math and foreign language than to other biology courses. We acknowledge that each section summarizes and simplifies a significant body of literature and encourage readers to pursue a deeper understanding of pedagogy elsewhere.\n\nExample-based learning has a long history in teaching coding, and consists of the instructor providing examples of how to solve a given problem using code. Sometimes, these examples are static and provided in a textbook or on slides. Other forms of example-based learning require the learners to actively engage with the code. An especially effective way to teach computational biology is to combine multiple forms of example-based learning (Renkl, 2014a). For example, an instructor might use all three methods detailed below: first show learners some written examples, perhaps in pre-reading for a class, then use live coding during class and have the learners follow along, and subsequently have learners complete a series of examples with increasing amounts of code that learners need to fill in during in-class problem sets or in homework assignments.\n\nWritten examples. Written examples are often a good starting point to introduce learners to the concepts and structure of solutions. Written tutorials allow learners to go at their own pace. This ensures learners take the time they need. However, learners frequently skim over static written examples or skip formative assessment questions, confident that they understand what the code is doing. Students may need to be required to critically evaluate the code to truly ensuring their understanding. For this reason, written examples are most effective when integrated with other example-based learning methods (Renkl, 2014a).\n\nLive coding. Live coding is a form of example-based learning in which learners watch the instructor solve problems and in most cases the learners follow along. The act of typing along forces learners to actively enter code, which is a hands-on version of a worked example. This means the instructor must proceed at a reasonable pace that learners can follow. The instructor can stop and ask learners challenge questions as formative assessment to ensure that learners apply their knowledge immediately and question their own understanding. The instructor can also set up a problem, and pause to have learners figure out the rest of the exercise. This live coding approach has been popularized by Data Carpentry and similar coding workshops. Live coding can help teach aspects of programming that are not easily taught by viewing static code, such as incremental coding (writing a few lines and testing them) and debugging. From the perspective of the learners, live coding is generally preferable to viewing static code, especially when learners are able to code along with instructors (Raj et al., 2018).\n\nDisadvantages of live coding include that everyone goes at the same pace. This may be too fast for some and too slow for others. One possibility to alleviate these disadvantages is to flip the classroom and provide learners with pre-recorded live-coding experiences. The learners then come to class where they complete an exercise or a series of faded examples.\n\nFaded examples. Faded examples are a form of example-based learning that can be more effective than static worked examples (Schwonke et al., 2009). Faded examples include sets of worked examples, beginning with a complete example. With each subsequent example, key problem solving steps are removed and learners must insert the steps themselves (Renkl, 2014b). Faded examples have been demonstrated to require less learning time with potentially better learning outcomes (Schwonke et al., 2009).\n\nIn the context of teaching computation to biologists, faded examples are particularly facilitated by servers. For example, a Jupyter notebook containing faded examples may be made available to learners on the server. If learners successfully complete the code they will produce an expected outcome, thus allowing for the learner to self-correct their work. Fillable Jupyter notebooks using the faded examples technique force learners to engage with the material and actively problem-solve. Fillable notebooks are particularly useful because they run the code and allow the learner to immediately discover if they produced the right answer. This type of immediate feedback allows the learner to work towards the correct answer, rather than turning in work that they believe to be correct and receiving a low grade on something they put time and effort into. An important component is providing learners with a solution set after they have had the opportunity to solve the faded examples so that they can see where they went wrong if they are unsuccessful.\n\nOne of the main skills underlying learning to code is learning how to solve problems. Writing code can be viewed as a method of communicating to the computer the precise steps of the analysis. In fact, code serves as a written record of the analytical steps, and is one that is instantly reproducible. When the learning outcomes are not necessarily tied to learners having full mastery of a specific language’s syntax, shorthand notation can be used to outline the steps the learner would take to get to the end result, removing the added layer of correct syntax and highlighting the importance of the problem-solving elements. This method, called pseudocode, can help learners logically scaffold their thought process, and therefore their code, regardless of which language they will be coding in. Learners can be required to write the solution to their problem in pseudocode to demonstrate their thought process, and then use that pseudocode to write their code (e.g., Olsen, 2005). Courses may rely on pseudocode during timed exams to relieve exam stress. Note, however, that if learners are expected to interpret or elaborate on pseudocode — especially in an exam setting — that the meaning behind each pseudocode term is clearly and unambiguously defined (Cutts et al., 2014).\n\nWorking in groups or pairs can result in numerous positive outcomes in the biological sciences, including enhancing scientific thinking and attitudes about biology, aiding in the instruction and evaluation of course material, providing a better understanding of practical skills and their applicability in real-world environments, improving reading, writing, and social skills, and supporting learning for a broader array of learners (reviewed in Lord, 2001). In the context of teaching computational biology, group projects and other forms of cooperative learning have frequently been used to facilitate example-based learning (e.g., Emery & Morgan, 2017; Fuselier et al., 2011; Korcsmaros et al., 2013). One creative cooperative learning teaching tool for computational biology is pairing learners up and having one student be the ‘driver’, who types in the code, and one be the ‘navigator’, who has to tell the driver what to type. This exercise forces learners to think carefully about the process of creating code. Even if learners are not expected to work through examples or produce a project in pairs or groups, cooperative learning can still be implemented by facilitating discussion and collaboration among learners. This can be done in class (e.g., through think-pair-share exercises, in which learners are encouraged to think about a problem individually, then discuss in pairs or small groups) or outside of class through discussion forums. For instance, learners could be encouraged or required to participate in discussions on the course website (e.g., Blackboard/Moodle) or a Slack workspace created for the class, or to do peer review of other learners’ projects. Learning from each other can be a powerful way to get learners engaged with the course work, and can solidify their understanding by having them teach each other (Treisman, 1992).\n\nHowever, as most students and instructors know, group work can create problems and strife. Interpersonal issues within pairs or groups can hinder the learning process. Sometimes groups or pairs are unbalanced with regard to prior knowledge, speed of learning, or effort contributed to the project. Additionally, group size or composition can lead to an entire group struggling to succeed (e.g., Compeau, 2019). Therefore, although group projects are frequently used in computational biology and can be a useful tool, relying only on group-based projects for critical assessments in a course may not be the best solution.\n\nIncorporating computational biology into the classroom comes with additional challenges that may not be present in other biology courses. Many learners will have some prior biological knowledge but have no experience with computer coding. This creates a situation where learners have many questions but often feel out of their comfort zone, or are simply uninterested in the computational components of the course material. These factors combine result in a number of different challenges for instructors to help learners successfully navigate the computational side of a biology course.\n\nDemonstrating value. The unfamiliarity of coding can sometimes lead learners to push back against the idea of learning to code. In our experience, demonstrating how coding can be a useful tool for doing the same analysis multiple times (e.g., generate the same type of plot for different datasets) helps learners see the value in learning to code. This demonstration is especially powerful when the learners are enabled to discover the utility of coding on their own. For example, an instructor could provide students with a large dataset and encourage the learners to try basic analyses (e.g., producing summary statistics or basic plots) in both R and Excel. Particularly when learners have not opted into learning computational skills — for example, when computational skills are incorporated into required biology classes — showing learners the value of saving future time and effort is very important.\n\nTeaching how to ask for help. Not all learners will automatically ask for help (see Box 3). As instructors, we need to encourage learners to seek our help when they are struggling. One method we have found successful is to provide learners with rewards for asking for help, such as an automatic extension on deadlines if learners attend office hours. Having knowledgeable teaching assistants or support staff to circulate the classroom and check on learner progress during in-class exercises is also helpful (see Box 3 for further notes on asking for help). In these cases, instructing the teaching support staff to first approach learners who do not have their hands raised will provide help to a larger proportion of the class, especially many learners who need the most help. To help learners signal that they need help, instructors can use a system that does not force students to stand out by raising their hands. For instance, Data Carpentry uses post-it notes to discreetly signal to instructors when learners need assistance. Incorporating live coding into lectures is another way to normalize asking for help; while live coding, instructors can demonstrate that errors are a natural part of coding, how to effectively search for solutions to problems, and how to look up syntax details.\n\nAsking for help is critical for learners to be able to overcome confusion, solidify their skills, and deepen their knowledge and understanding. However, cultural differences exist that may prevent learners from seeking help, even when they need it most. Universities in particular have the unspoken expectation that undergraduates will seek help when they need it, but people from some backgrounds will not necessarily know this, or may feel uncomfortable approaching their teachers, who are in positions of authority (Jack, 2016). Different cultures may perceive asking questions in class in different ways; some cultures view questions as a way to gain recognition, whereas others view questions as a demonstration of ignorance. The latter group are less likely to ask questions in class and thus do not receive either needed help or engagement with the instructor.\n\nLarge class sizes. For instructors who are looking to incorporate computational biology into large classes, such as in an introductory biology course, the prospect of testing and grading code turned in as assignments may be daunting. We have found that relying on knowledgeable TAs, support staff, and demonstrators is the best way to ensure that all learners receive the help that they need. Placing learners in pairs or small working groups (see the section on Cooperative Learning above) can also provide learners with support and feedback that will facilitate their learning, without placing the entire burden on instructors and TAs. Additionally, relying on IT support to help manage the computing platforms can reduce the workload for the instructors. Furthermore, using assignments that can be automatically graded (e.g., mimir classroom) will help reduce the time teachers must spend grading.\n\nPlagiarism. Writing code is more similar to math than to writing, as there is a correct answer (or perhaps several correct answers), and it can be difficult to detect whether learners arrived at that answer independently or through copying someone else’s solution. Plagiarism can be exacerbated by the common phenomenon of searching for solutions to a given problem online and copying or adapting posted solutions without attributing proper credit (Gaspar & Langevin, 2007). Some of the programs that grade code include plagiarism detection components (Pears et al., 2007), which can help instructors identify when plagiarism has occurred. One approach to ensure learners understand code is to require commented explanation of each component of their code, and ensure these comments are different for each learner. Requiring unique comments from learners has the advantage that it allows learners to be able to search for the functions and arguments to functions that are needed to implement their analytical work, which is common practice when writing code. Another approach is to allow learners to work on assessments as a group, but have each learners write out their own pseudocode explanations of the code. Instructors are encouraged to discuss issues surrounding intellectual property and plagiarism and reinforce the importance of citing sources.\n\n\nConclusions\n\nComputing is increasingly being incorporated into coursework at all levels in biology. Computing is both a key skill for learners and supports additional learning by enabling the incorporation of data and analysis into classrooms. Instructors have a range of choices to deliver computational lessons. From the computing platform to how the instructor delivers the lesson, every aspect of a computational classroom can shape learners’ knowledge and confidence. In this paper, we have outlined the costs and benefits of several popular computing platforms. Given the variety of options, instructors should be able to find a platform that best fits their course learning objectives, available resources, class size, and learner backgrounds. One important consideration in this decision is how the platform of choice affects learner access to course content. Many of these computing platforms can support inclusion in the classroom (and thus, ultimately, in fields and careers that incorporate computation) by providing access to the same technologies to all learners. We have also discussed several teaching strategies for computational biology and other courses with computation, with the goal of improving student engagement, learning, and retention.\n\n\nData availability\n\nThere are no data associated with this article",
"appendix": "Acknowledgments\n\nLinda Forrester contributed significant time to implementing labs using R in URI’s Introductory Biology labs, and Chi Shen provided support for URI’s R Studio Server, on which some of this work is based. This manuscript is a product of discussions from iEvoBio’s 2019 annual meeting.\n\n\nReferences\n\nBarone L, Williams J, Micklos D: Unmet needs for analyzing biological big data: A survey of 704 NSF principal investigators. PLoS Comput Biol. 2017; 13(10): e1005755. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–1423. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCompeau P: Establishing a computational biology flipped classroom. PLoS Comput Biol. 2019; 15(5): e1006764. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCook J, Bedford D, Mandia S: Raising climate literacy through addressing misinformation: Case studies in agnotology-based learning. Journal of Geoscience Education. 2014; 62(3): 296–306. Publisher Full Text\n\nCummings MP, Temple GG: Broader incorporation of bioinformatics in education: opportunities and challenges. Brief Bioinform. 2010; 11(6): 537–43. PubMed Abstract | Publisher Full Text\n\nCutts Q, Connor R, Michaelson G, et al.: Code or (not code): Separating formal and natural language in CS education. In Proceedings of the 9th Workshop in Primary and Secondary Computing Education WiPSCE ’ 14, ACM Berlin, Germany. 2014; 20–28. Publisher Full Text\n\nEmery LR, Morgan SL: The application of project-based learning in bioinformatics training. PLoS Comput Biol. 2017; 13(8): e1005620. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFaust JL, Paulson DR: Active learning in the college classroom. Journal on Excellence in College Teaching. 1998; 9: 3–24. Reference Source\n\nFuselier L, Bougary A, Malott M: From trace evidence to bioinformatics: putting bryophytes into molecular biology education. Biochem Mol Biol Educ. 2011; 39(1): 38–46. PubMed Abstract | Publisher Full Text\n\nGaspar A, Langevin S: Restoring \"coding with intention\" in introductory programming courses. In Proceedings of the 8th ACM SIGITE Conference on Information Technology Education SIGITE ’ 07 ACM Destin, Florida, USA. 2007; 91–98. Publisher Full Text\n\nGibson JP, Mourad T: The growing importance of data literacy in life science education. Am J Bot. 2018; 105(12): 1953–1956. PubMed Abstract | Publisher Full Text\n\nGoff SA, Vaughn M, McKay S, et al.: The iPlant Collaborative: Cyberinfrastructure for Plant Biology. Front Plant Sci. 2011; 2: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuerta-Cepas J, Serra F, Bork P: ETE 3: Reconstruction, Analysis, and Visualization of Phylogenomic Data. Mol Biol Evol. 2016; 33(6): 1635–1638. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJack AA: (No) harm in asking: Class, acquired cultural capital, and academic engagement at an elite university. Sociology of Education. 2016; 89(1): 1–19. Publisher Full Text\n\nKember D, Ho A, Hong C: The importance of establishing relevance in motivating student learning. Active Learning in Higher Education. 2008; 9(3): 249–263. Publisher Full Text\n\nKjelvik MK, Schultheis EH: Getting Messy with Authentic Data: Exploring the Potential of Using Data from Scientific Research to Support Student Data Literacy. CBE Life Sci Educ. 2019; 18(2): es2. PubMed Abstract | Publisher Full Text\n\nKluyver T, Ragan-Kelley B, Pérez F, et al.: Jupyter notebooks – a publishing format for reproducible computational workflows. In Positioning and Power in Academic Publishing: Players, Agents and Agendas. (F. Loizides and B. Schmidt, eds.) IOS Press. 2016; 87–90. Publisher Full Text\n\nKorcsmaros T, Dunai ZA, Vellai T, et al.: Teaching the bioinformatics of signaling networks: an integrated approach to facilitate multi-disciplinary learning. Brief Bioinform. 2013; 14(5): 618–632. PubMed Abstract | Publisher Full Text\n\nLoman NJ, Quinlan AR: Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics. 2014; 30(23): 3399–3401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoman N, Watson M: So you want to be a computational biologist? Nat Biotechnol. 2013; 31(11): 996–998. PubMed Abstract | Publisher Full Text\n\nLord TR: 101 reasons for using cooperative learning in biology teaching. The American Biology Teacher. 2001; 63(1): 30–38. Publisher Full Text\n\nMcKinney W: Data structures for statistical computing in python. In Proceedings of the 9th Python in Science Conference. (S. van der Walt and J. Millman, eds.). 2010; 51–56. Reference Source\n\nMerchant N, Lyons E, Goff S, et al.: The iPlant Collaborative: Cyberinfrastructure for Enabling Data to Discovery for the Life Sciences. PLoS Biol. 2016; 14(1): e1002342. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller MA, Pfeiffer W, Schwartz T: Creating the cipres science gateway for inference of large phylogenetic trees. In 2010 gateway computing environments workshop (GCE) Ieee. 2010; 1–8. Publisher Full Text\n\nOlsen AL: Using pseudocode to teach problem solving. Journal of Computing Sciences in Colleges. 2005; 21(2): 231–236. Reference Source\n\nParadis E, Schliep K: ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2018; 35(3): 526–528. PubMed Abstract | Publisher Full Text\n\nPears A, Seidman S, Malmi L, et al.: A survey of literature on the teaching of introductory programming. In Working Group Reports on ITiCSE on Innovation and Technology in Computer Science Education ITiCSE-WGR ’ 07 ACM Dundee, Scotland. 2007; 204–223. Publisher Full Text\n\nRaj AGS, Patel JM, Halverson R, et al.: Role of live-coding in learning introductory programming. In Proceedings of the 18th Koli Calling International Conference on Computing Education Research ACM. 2018; 13. Publisher Full Text\n\nRenkl A: Learning from worked examples: how to prepare students for meaningful problem solving. In Applying Science of Learning in Education: Infusing Psychological Science into the Curriculum. (V. A. Benassi, C. E. Overson, and C. M. Hakala, eds.). Society for the Teaching of Psychology. 2014a; 118–130. Reference Source\n\nRenkl A: Toward an instructionally oriented theory of example-based learning. Cogn Sci. 2014b; 38(1): 1–37. PubMed Abstract | Publisher Full Text\n\nRStudio Team: RStudio: Integrated Development Environment for R. RStudio, Inc. Boston, MA. 2015.\n\nRStudio Team: RStudio Server Professional Edition 1.2.5001-3. RStudio, Inc. Boston, MA. 2019. Reference Source\n\nSchwonke R, Renkl A, Krieg C, et al.: The worked-example effect: Not an artefact of lousy control conditions. Comput Human Behav. 2009; 25(2): 258–266. Publisher Full Text\n\nStefik A, Siebert S: An empirical investigation into programming language syntax. ACM T Comput Educ. 2013; 13(4): 19. Publisher Full Text\n\nStewart CA, Cockerill TM, Foster I, et al.: Jetstream: a self-provisioned, scalable science and engineering cloud environment. In: Proceedings of the 2015 XSEDE Conference: Scientific Advancements Enabled by Enhanced Cyberinfrastructure ACM. 2015; 29. Publisher Full Text\n\nSukumaran J, Holder MT: DendroPy: a Python library for phylogenetic computing. Bioinformatics. 2010; 26(12): 1569–1571. PubMed Abstract | Publisher Full Text\n\nTowns J, Cockerill T, Dahan M, et al.: Xsede: accelerating scientific discovery. Comput Sci Eng. 2014; 16(5): 62–74. Publisher Full Text\n\nTreisman U: Studying students studying calculus: A look at the lives of minority mathematics students in college. Coll Math J. 1992; 23(5): 362–372. Publisher Full Text\n\nWilliams J, Drew J, Galindo-Gonzalez S, et al.: Barriers to integration of bioinformatics into undergraduate life sciences education. BioRxiv. [Preprint]. 2017. 204420. Publisher Full Text\n\nWilson Sayres MA, Hauser C, Sierk M, et al.: Bioinformatics core competencies for undergraduate life sciences education. PLoS One. 2018; 13(6): e0196878. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu Q, Anderson JR: Problem-solving transfer among programming languages. Tech rep. Carnegie-Mellon University Pittsburgh PA Artificial Intelligence And Psychology. 1990. Reference Source"
}
|
[
{
"id": "56230",
"date": "26 Nov 2019",
"name": "Tiffany Timbers",
"expertise": [
"Reviewer Expertise Data Science",
"Biology/Neuroscience"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a nice introductory overview of teaching computational biology. They cover several key practical and pedagogical aspects one needs to consider when designing and teaching a computational biology course. These include choice of programming language, choice of computing platform, and best practices for teaching computational materials. This review will serve as a good overview of the field at the present time for newcomers.\n\nBelow I suggest some things that could improve the manuscript:\nIn the introduction, where the rationale for teaching computation in biology classes is explained. I suggest adding an additional rationale: to give students the tools, skills and motivation to create analyses that are reproducible and transparent, regardless of the size of the data set (\"it's worth it even for small data sets).\nI agree that Software and Data Carpentry workshops are great places to start to get computational skills, however to learn the skills to teach computation to others, faculty will need additional training. For example the Software and Data Carpentry Instructor training courses (perhaps this is what you were referring to?). Another resource that is aligned with this is \"Teaching Tech Together\" by Greg Wilson.\nIn the \"Choice of computing language\" section, the two paragraphs comparing R and Python could be improved. For example, both R and Python now have easy to use tools for working with rectangular data, but that is not made clear in the Python paragraph. The authors do write that \"Pandas can be used for teaching foundational data skills\", but someone unfamiliar with Pandas will not likely make the leap that manipulating rectangular data is what is meant here. Essentially, for simple, rectangular data tasks, that do not involve complex statistics, both R and Python are excellent language choices. And one should pick the one that their colleagues and collaborators use. However, when analysis tasks get more specific, then one might have to use the language where the package exists with specific tools they need.\n\nIn the first paragraph of the \"Cloud-based computing platforms\" section, I think it is important to communicate that R (and other languages) can be used with Jupyter. This is stated later, but R readers might skip the Jupyter section later on if this is not communicated earlier. Also, I would remove the sentence \"Jupyter supports the R language. However, the interface for R in Jupyter is different than RStudio, a dominant way to interact with R\". Jupyter is getting better and better at working with R, and thus if only a notebook is needed, there is no reason not to use Jupyter with R. However, if your code project is more complex and needs/benefits from a real IDE, then of course RStudio should be used (and the same argument goes for Python).\nIn the \"Teaching methods\" section attribution is given to Data Carpentry for popularizing the live coding approach of teaching coding. However, Software Carpentry proceeded this. This also happens again later in the manuscript when the post-it note system is discussed. In earlier parts of this manuscript, Software Carpentry is referred to alone. Perhaps it would be better to refer to these two organizations together consistently throughout the manuscript as the \"Software and Data Carpentry organizations\" (or something like that).\n\nThere are several other great coding exercise strategies that would be great to point out in this article (Parson's problems, tracing (execution and values), minimal fix, refactoring, etc). These are described quite well in \"Teaching Tech Together\" by Greg Wilson.\nOne last addition I might add to the platform and/or teaching method section is the potential for the use of autograding and/or automated feedback using tests in computational biology. When giving students a computing challenge, tests can also be provided to see if the student's code creates an object or function with the desired properties or functionality. In Jupyter notebooks, these tests can be paired with tools such as nbgrader or OK for autograding. This is being used in several undergraduate Data Science courses at several universities (e.g., Berkley & the University of British Columbia, etc).\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": "57707",
"date": "20 Jan 2020",
"name": "Thomas Kluyver",
"expertise": [
"Reviewer Expertise Academic background in biology",
"since transitioned into scientific computing"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article gives an overview of different available options and considerations for adding a programming component to a university-level biology syllabus. The authors don't give a one-size-fits-all answer, choosing quite reasonably to highlight where different tools make most sense. I appreciated the callouts highlighting issues that could affect some students more than others.\nIn the context of teaching with Jupyter notebooks, I would mention nbgrader, an open source tool which works with JupyterHub to support assignments in notebooks, with semi-automated grading.\nI think the article could be made clearer by using the word 'cloud' more carefully. Admittedly marketing has made the term extremely nebulous in general, but it's used here with two distinct meanings: first for providing software on a central server or cluster, and then more specifically for commercial services which rent computing resources.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/8-1854
|
https://f1000research.com/articles/8-1875/v1
|
07 Nov 19
|
{
"type": "Research Article",
"title": "Peptide arrays of three collections of human sera from patients infected with mosquito-borne viruses",
"authors": [
"Maria del Pilar Martinez Viedma",
"Nurgun Kose",
"Leda Parham",
"Angel Balmaseda",
"Guillermina Kuan",
"Ivette Lorenzana",
"Eva Harris",
"James E. Crowe Jr.",
"Brett E. Pickett",
"Maria del Pilar Martinez Viedma",
"Nurgun Kose",
"Leda Parham",
"Angel Balmaseda",
"Guillermina Kuan",
"Ivette Lorenzana",
"Eva Harris",
"James E. Crowe Jr."
],
"abstract": "Background: Global outbreaks caused by emerging or re-emerging arthropod-borne viruses (arboviruses) are becoming increasingly more common. These pathogens include the mosquito-borne viruses belonging to the Flavivirus and Alphavirus genera. These viruses often cause non-specific or asymptomatic infection, which can confound viral prevalence studies. In addition, many acute phase diagnostic tests rely on the detection of viral components such as RNA or antigen. Standard serological tests are often not reliable for diagnosis after seroconversion and convalescence due to cross-reactivity among flaviviruses. Methods: In order to contribute to development efforts for mosquito-borne serodiagnostics, we incubated 137 human sera on individual custom peptide arrays that consisted of over 866 unique peptides in quadruplicate. Our bioinformatics workflow to analyze these data incorporated machine learning, statistics, and B-cell epitope prediction. Results: Here we report the results of our peptide array data analysis, which revealed sets of peptides that have diagnostic potential for detecting past exposure to a subset of the tested human pathogens including Zika virus. These peptides were then confirmed using the well-established ELISA method. Conclusions: These array data, and the resulting peptides can be useful in diverse efforts including the development of new pan-flavivirus antibodies, more accurate epitope mapping, and vaccine development against these viral pathogens.",
"keywords": [
"peptide arrays",
"mosquito-borne viruses",
"Zika virus",
"serodiagnostic",
"bioinformatics",
"B-cell epitopes"
],
"content": "Introduction\n\nZika virus (ZIKV) is an arbovirus within the Flavivirus genus and the Flaviviridae family. In addition to ZIKV, many other mosquito-borne viruses exist that negatively affect public health, including dengue virus (DENV) and chikungunya virus (CHIKV), among others. ZIKV is primarily transmitted by the bite of infected Aedes spp. mosquitoes, with limited instances of sexual transmission also being reported1–4. The recent worldwide epidemic has demonstrated that ZIKV is a neuropathic virus that is associated with fetal microcephaly and other congenital defects in infected pregnant women, and Guillain-Barré syndrome in adults5. Due to the number of ZIKV infections in recent years and the continued threat of ZIKV re-emerging around the world, there is still an urgent need for rapid and accurate surveillance assays in order to rapidly identify new outbreaks. Distinguishing between infection with multiple co-circulating arboviruses that have similar clinical signs and symptoms makes accurate prevalence calculations and diagnosis extremely difficult—especially after convalescence6–10.\n\nThe sequence similarity at the amino acid level in many flavivirus immunogenic protein regions contributes to the observed cross-reactivity in serological assays, which is especially high in the E protein and also present in the NS1 protein11. Although reports showing antibodies against other viral proteins are detectable12, the E and NS1 proteins are the primary targets of the humoral anti-flavivirus immune response in humans13–15.\n\nRecent efforts to generate whole-genome sequences for these pathogens enable the application of bioinformatics tools to mine the data for trends and patterns that can be clinically applicable16–20. The meta-CATS (metadata-driven Comparative Analysis Tool for Sequences) algorithm is a statistical workflow that rapidly identifies sequence variations that significantly correlate with the associated metadata for two or more groups of sequences21. This algorithm has been used previously to identify residues within 15-mer linear peptide regions that have high predicted specificity and sensitivity values and that could therefore be useful for detecting antibodies against a variety of mosquito-borne Flavivirus species22. Quantifying the reactivity of this set of peptides using high-throughput custom peptide arrays enables the efficient and simultaneous testing of the set of peptides against a variety of serum samples with higher efficiency than what is possible with manual enzyme-linked immunosorbent assay (ELISA) technology alone23.\n\nThe data presented here are the product of combining upstream computational methods to predict peptides capable of distinguishing each virus with downstream high-throughput screening of relevant sera using peptide arrays. We have recently completed an analysis of 137 serum samples using custom peptide arrays (each containing 866 experimental viral peptides) to identify 15-mer linear peptides that could be useful as serodiagnostic reagents to detect prior infection with mosquito-borne viruses. Specifically, we tested peptides representing different co-circulating mosquito-borne viruses, including: ZIKV, DENV 1–3, CHIKV and West Nile virus (WNV). Applying machine learning, a weighting scheme, and B-cell epitope prediction algorithms to these data enabled us to identify pools of 8–10 peptides that are predicted to be immunodominant across human sera from previously infected individuals in Central and South America. In addition, we have separately evaluated these peptides using an ELISA method with a set of well-characterized sera. These data could be used by the scientific community to develop improved serological diagnostic methods for detecting past infection with one or more of these viral pathogens.\n\n\nMethods\n\nA subset of the previously predicted diagnostic peptides21, representing multiple mosquito-borne virus species and subtypes, were synthesized at the Center for Protein and Nucleic Acid Research at The Scripps Research Institute (TSRI)23,24. This selected collection of peptides consisted of 15-mers with sequences that represented the consensus amino acid sequence among strains belonging to each of our six target taxa including: CHIKV, DENV1, DENV2, DENV3, WNV, and ZIKV. Peptides on the array that represented mosquito-borne virus taxa for which there were no serum samples were ignored in downstream quantification and computation. As such, a total of 25, 51, 28, 34, or 70 peptides in the E protein as well as 15, 19, 15, 23, or 70 peptides in the NS1 protein (all derived from DENV1, DENV2, DENV3, WNV, or ZIKV sequences, respectively) were evaluated in these experiments. A set of 25 peptides spanning portions of the CHIKV E2 protein that had previously been reported as relevant for detecting anti-CHIKV antibodies were also included25. Synthesized peptides were suspended in 12.5 μL DMSO and 12.5 μL of ultra-pure water. Immediately prior to printing, suspended peptides were diluted 1:4 in a custom protein printing buffer [saline sodium citrate (SSC): 300 mM sodium citrate, pH 8.0, containing 1 M sodium chloride and supplemented with 0.1% Polyvinyl Alcohol (PVA) and 0.05% Tween 20], in a 384-well non-binding polystyrene assay plate. Two positive control peptides, hemagglutinin A (HA) (YPYDVPDYA) and FLAG tag (DYKDDDDK), together with a dye that permanently fluoresces at 488 nm (Alexa Fluor 488) were included in the print to guide proper grid placement and peptide alignment, as well as to serve as printing controls as well as controls to quantify the maximum fluorescence for the assays.\n\nQuadruplicate sets of all peptides were printed onto N-hydroxysuccinimide ester (NHS-ester) coated NEXTERION Slide H (Applied Microarrays) slides at an approximate density of 1 ng/spot, using a Microgrid II (DigilabGlobal) microarray printing robot equipped with solid steel (SMP4, TeleChem) microarray pins. Humidity was maintained at 50% during the printing process. Immediately prior to interrogating the arrays, slides were blocked for 1 h with ethanolamine buffer to quench any unreacted NHS-ester on the slide. All slides were used within 2 months of printing and were stored at -20°C23.\n\nSpent diagnostic serum samples were provided by collaborators working under three separate clinical studies in Honduras, the United States, and Nicaragua. These sera were collected from a total of 137 consented human patients under IRB supervision and were characterized as positive for antibodies against at least one of: ZIKV, DENV1, DENV2, DENV3, WNV, and/or CHIKV.\n\nA total of 32 deidentified plasma samples from patients suspected of Zika, chikungunya or dengue in Honduras were obtained at the discretion of health care providers at the Hospital Escuela Universitario from patients (ages 6–73 years old). These samples were sent to the Centro de Investigaciones Geneticas at the Universidad Nacional Autonoma de Honduras in Tegucigalpa, Honduras for ZIKV, CHIKV and/or DENV molecular testing. Of these patients, 23 had infection with DENV and nine had infection with ZIKV confirmed by RT-qPCR during the acute phase. Convalescent samples were collected from these patients 10–30 days post-onset of symptoms between June 1 to November 30, 2016 and were tested on the custom arrays.\n\nA total of 73 de-identified human serum samples were obtained from the Vanderbilt Vaccine Center Biorepository. Sera from individuals with previous history of natural infection with DENV, WNV, CHIKV, or ZIKV (confirmed by serology or RT-qPCR) while traveling in the Caribbean, Central or South America, or West Africa were included on arrays. For WNV, sera were from individuals with confirmed previous history of natural infection contracted during an outbreak in 2012 in Dallas, TX. The samples were collected in the convalescent phase, months to years after post-onset of symptoms.\n\nA total of 32 de-identified human sera were collected from the Pediatric Dengue Cohort Study in Managua, Nicaragua26,27. Early convalescent-phase samples were collected 15–17 days post-onset of symptoms from 9 Zika cases that were confirmed as positive for ZIKV infection by real-time RT-qPCR between January and July, 2016. Late convalescent samples were obtained from 21 DENV-positive cohort participants after RT-qPCR confirmed DENV1 (n=7), DENV2 (n=8), or DENV3 (n=6) infection and 2 DENV-negative subjects, all in 2004–2011, prior to the introduction of ZIKV to Nicaragua. Samples were analyzed by inhibition ELISA28,29 and neutralization assay30,31. The PDCS was approved by the IRBs of the University of California, Berkeley, and Nicaraguan Ministry of Health. Parents or legal guardians of all subjects provided written informed consent; subjects 6 years old and older provided assent.\n\nOnce the peptide microarrays were printed, aliquots from a subset of samples were used to optimize the screening and detection processes. Specifically, dilutions ranging from 1:50 to 1:1000 were evaluated to determine the optimal dilution level for subsequent screening. A 1:200 dilution was selected to achieve an optimal balance between the available aliquot volumes and assay sensitivity.\n\nThe 137 characterized sera were separately subjected to high-throughput screening using the synthesized peptide arrays. Sera were tested for IgG reactivity using the custom peptide array at TSRI. For immunolabeling, the incubation area around the printed grids was circumscribed using a peroxidase anti-peroxidase (PAP) hydrophobic marker pen (Research Products International Corp) and the subsequent steps were performed in a humidified chamber at room temperature on a rotator. Control anti-HA (mAb 12CA5, Scripps Research, mouse IgG, RRID:AB_514505) and anti-FLAG monoclonal antibodies (Invitrogen, MA1-142-A488, RRID:AB_2610653) were assayed at a concentration of 10 μg/ml while 10 μl of human sera were diluted 1:200 in PBS buffer containing Tween (PBS-T) and incubated for 1 h followed by three washes in PBS buffer. The arrays were then incubated for 1 h with goat anti-human IgG tagged with Alexa Fluor® 488 (Invitrogen, cat. #: A-11013, RRID: AB_2534080) as a secondary antibody. Arrays were washed three times in PBS-T, two times in PBS, and another two times in deionized water and centrifuged to dry at 200 × g for 5 mins.\n\nThe fluorescence of the processed slides was quantified using a ProScanArray HT (Perkin Elmer) microarray scanner at 488 nm and 600 nm, and images were saved as high-resolution TIF files. Imagene® 6.1 microarray analysis software (BioDiscovery; ImageJ could be used as an open-access alternative) was used to calculate the fluorescence intensity of the area within the printed diameter of each peptide as well as the fluorescence of the same diameter directly outside of the area occupied by each peptide. The mean and median fluorescence signal and background pixel intensities, as well as other data for each antigen, spot were calculated, digitized, and exported as individual rows in a comma-delimited file for subsequent analysis.\n\nA custom script32 was written to implement a previously described array processing workflow24 with a minor change to use the median foreground and background values instead of mean values to minimize outlier effects (available on GitHub). Negative background values were interpreted as zeroes. Briefly, background correction was calculated by subtracting the median background from the median foreground measurements for each spot on each array. Normalization was performed by dividing the background-corrected values for each spot on each slide by the non-control spot having the largest fluorescence value on each slide as has been described previously24. The quadruplicate spots for each peptide on each array were then summarized into a single value by calculating the median value of the quadruplicate spots for each peptide to further reduce the effects of any outliers. The normalized relative fluorescence intensity values for all peptides and all samples were output as a separate file together with summarized quantitative values indicating how well each peptide was recognized by each of the polyclonal serum samples.\n\nA separate script was used to transform all relative fluorescence intensity values for each peptide into Z-scores, and separate tables were constructed to contain the summarized Z-score values for all peptides (as columns) representing each of the viral taxa and all samples (as rows) that were tested with the peptide array. A random forest algorithm (randomForest version 4.6-12 package in R) was applied to each of these tables in order to identify the peptides that were best able to differentiate between each of the viral taxa. In this case, the number of trees generated in the random forest for each species was 100,000, and the number of variables randomly sampled as candidates at each split was equal to the square root of the number of columns present in each table.\n\nThe values representing the mean decrease in Gini index were calculated separately for samples obtained from each of the three collections as well as all possible combinations of two or more collections. These data were then used to identify the top 30 peptides according to their usefulness in identifying the correct virus taxon. The BepiPred algorithm was then used to predict the number of residues that are frequently present in B-cell epitopes, and would therefore contribute to increased affinity and binding by antibodies in downstream assays33. The peptides were then assigned a cumulative rank based on the epitope prediction and Gini values, and the 10 highest-ranking peptides across the E and NS1 proteins for each viral taxon, as well as 8 peptides in the E2 region for CHIKV, were categorized as the most likely to have high immunodominance and therefore be recognized by antibodies in sera collected from previously infected patients in the western hemisphere. Statistical comparisons of quantitative differences between the Gini and normalized fluorescence values for sets of peptides were performed using Student’s t-test.\n\nEach peptide was synthesized (LifeTein, LLC) and 2 ng of peptide was diluted in 50 μL of ddH2O. Natural human IgG protein (abcam, cat. # ab91102), complement component C1q from human serum (sigma, cat. #: C1740), and labelled secondary antibody (ThermoFisher, cat. #: A18817, RRID: AB_2535594) were used as additional controls. Pools of two peptides were used to coat duplicate wells on a 96-well Immulon 4HBX plate (ThermoFisher, cat. # 3855) and incubated at 4°C overnight. Next, 100 μL of blocking buffer (PBS+5% BSA) was added to each well and incubated for 2 hours at room temperature prior to three washing steps with washing buffer (PBS + 0.05% Tween 20). Human serum was diluted 1:25 in blocking buffer and 50 μL of this solution was added to each well prior to incubation for 2 hours at room temperature. Each plate was then washed four times with washing buffer and 50 μL of HRP-conjugated anti-human IgG antibody (ThermoFisher, cat. #: A18817, RRID: AB_2535594; 0.1 mg/mL diluted 1:20,000) was added to each well, followed by incubation at room temperature for 2 hours. Each plate was then washed four additional times before incubating at room temperature for 30 minutes with 75 μL of TMB substrate (abcam, cat. # ab171523). Then, 75 μL of stop solution (abcam, cat. # ab171529) was added to each well and a BioTek-synergy HT plate reader was used to quantify the fluorescence in each well at 450nm within 15 minutes.\n\nA normalization process was implemented that adjusted fluorescence values in each well based on the control wells included on each ELISA plate. This normalization enabled the downstream comparison between plates34. A downstream quality control method was also implemented to ignore results from ELISA plates that displayed high levels of background, inconsistent signal from multiple control wells, and samples observed to have at least two wells for each taxon with higher than expected signal.\n\nAll samples evaluated on the peptide arrays and ELISA plates were acquired from patients under informed consent and approved by the Ethical or Institutional Review Board at each participating institution, including: Universidad Nacional Autonoma de Honduras (IRB 00003070), Vanderbilt University (IRB 8675), University of California, Berkeley (Committee for Protection of Human Subjects 2010-09-2245), and the Comite Institutional de Revision Etica (NIC-MINSA/CNDR CIRE-09/03/07-008.ver19).\n\n\nResults\n\nOverall, we screened 137 unique serum samples for their reactivity against a panel of viral peptides (Figure 1 and Underlying data35). These samples, together with the clinical diagnosis, were collected from patients with known past exposure to at least one of the viruses targeted by our peptides (Table 1). Also contained within the Underlying data are files describing the metadata of peptides included on the array and each experimental sample35.\n\nA graphical depiction of the processes for predicting, screening, processing, and validating peptide array experiments.\n\nThe data from each array is contained in a single tab-delimited text file and contains the quantitative data captured from a single serum sample on a single peptide array35. A subset of the fields in each file include: location of each peptide spot on the array, peptide identifier, raw mean and median foreground fluorescence at 488 nm, raw mean and median background fluorescence at 488 nm, and other data collected from the raw image.\n\nA matrix containing the transformed Z-score values for each peptide was then formatted for input into a random forest (RF) machine learning algorithm to assist with ranking peptides according to virus taxon. To do so, a column was added to the matrix assigning each sample to the virus taxon that was known to have infected the patient (e.g. “Zika” or “Non-Zika”). Z-score values in columns containing the predicted peptides from each taxon were then captured and input into the RF algorithm.\n\nThe benefit of the RF algorithm is that it is capable of ranking the importance of features, which are peptides in this case, based on a known classification. The ranking is based on the mean decrease in Gini index, which is a value that quantifies node impurity. In other words, the higher the Gini index value, the more important the feature is in correctly identifying the virus taxon.\n\nIn order to account for geographical, genetic, and population-based factors, we computed the mean decrease in Gini index for individual collections (e.g. Nicaragua or Honduras), all relevant pairs of collections (e.g. Nicaragua and Honduras, Honduras and United States), and the combination of all collections from our sera providers. These calculations were accompanied by a class-error rate that quantifies the number of samples characterized as being positive for ZIKV that were predicted to be ZIKV samples.\n\nThis class-error rate information for each individual or combination of collections was then used to weight the peptide rankings results. Briefly, this involved multiplying the average rank for each peptide in each comparison by the average weight and dividing it by the sum of weight. This process works to increase the rank of peptides that have consistently high Gini values. We used these rankings to identify the top 25 species-specific peptides for each virus taxon. This process was repeated for non-ZIKV samples, including WNV, DENV1-3, and CHIKV.\n\nIn order to conserve resources for the peptide array and decrease the number of peptides that would be incorporated into the future ELISA assay, we used the existing BepiPred 2.0 algorithm to predict which of our 15-mer peptides contain the highest number of amino acids that are most often recognized by antibodies36. These B-cell epitope predictions were then used to reduce the 25 best peptides identified from machine learning, to the 10 best peptides that are predicted to not only be species-specific, but that are most likely to contain species-specific epitopes. In the case of ZIKV, we also reviewed the spot size and shape in the peptide array images to ensure that there were no irregularities that could negatively bias our results. The BepiPred 2.0 results enabled us to predict which peptides would be most seroreactive for each selected taxon. The mean maximum score from the BepiPred 2.0 analysis was calculated to be 0.58 (range: 0.55 – 0.63). These scores are associated with a specificity greater than 81%.\n\nGiven the serological cross-reactivity that has been reported among many of our targeted mosquito-borne viruses37, we recognized the need to validate the results of our high-throughput screen. To do so, we not only ensured that those generating the peptide array data were “blinded” to the phenotype of each sample, but we also computationally evaluated two distinct but complementary comparative and quantitative metrics that are described below.\n\nFirst, we compared two serum samples from pediatric patients that had not been infected with DENV prior to sample collection. The data from the DENV-specific peptides in these samples were then compared to those from a representative DENV-positive sample to verify the differences in signal between known positive and known negative samples. This comparison would also provide a better understanding of the contribution of cross-reactivity, which has been reported previously37, on our platform (Table 2). This comparison showed that the DENV-negative samples had less than four percent of the normalized fluorescence values, well below the 10 percent that was observed in the DENV-positive sample. Transforming these raw data into Z-scores further increases the observed differences in fluorescence values and, provides additional support to the unbiased nature of the data produced in these experiments.\n\n* Sample H22.\n\n** Sample H23.\n\n*** Sample H1.\n\nWe next wanted to assess the technical rigor of our approach by performing a statistical analysis of the observed experimental variation in the peptide array experiments. In this case, data was available for six of our target viruses for which sera was evaluated on the arrays. We specifically wanted to quantify the reactivity of the best-performing peptides for each sample against in a panel of comparisons (Table 3). The results from this analysis identified noticeable differences in the signals for ZIKV and WNV (Figure 2). However, we observed that the quantified values for the other four virus taxa were lower than the values for all samples combined and did not meet statistical significance when comparing known positive and negative samples (Figure 3). These results show that incorporating Gini scores and immune epitope predictions into our computational pipeline contributed to our ability to identify sets of peptides that were capable of distinguishing between past infection with a subset of our target viruses.\n\nA bar chart depicting the mean values of the best-performing peptides for samples that were characterized as: positive for the specified taxon, negative for the specified taxon, positive or negative for the specified taxon, and all samples across all viral taxa.\n\nA bar chart showing the -log10 p-value for the best-performing sets of peptides for each virus species. Comparisons include: samples that were positive for the virus vs. those that were negative for the virus (+ vs -), samples that were positive for the virus vs. all samples quantified for the virus), and samples that were positive vs. all quantified samples (+ vs. All). Values greater than 1.30 indicate p < 0.05.\n\nIt is also important to recognize that each peptide was printed at non-adjacent sites on each array in quadruplicate to minimize experimental bias due to the location of any given spot on the array. Incorporating technical replicates was an important component of the experimental design. Such an approach enables improved replication of the results and also increases the scientific rigor of the resulting dataset upstream of any data processing workflows.\n\nThe number of samples that were evaluated for prior exposure to each virus was insufficient to allow the use of in silico cross-validation techniques that are generally applied to the classifier predictions. We therefore designed custom 96-well ELISA plates to validate the ability of the peptides (Figure 4). The highest predicted reactivity to accurately detect prior infection by each of the target viruses.\n\nTesting of duplicate pairs of each peptide for each of the target virus taxa. Each column contains pairs of peptides that were predicted to be effective at differentiating each virus based on the bioinformatics processing of the peptide array data. (row A for ZIKV; row B for CHKV; row C for WNV; row D for DENV-1; row E for DENV-2; row F for DENV-3; row G for DENV-4; row H for YFV). Negative control wells, which were not coated with peptides are in column 12 and in wells with no label. Wells containing other control material are in column 11 (green) and include: C1q, naïve human immunoglobulin G, or only secondary antibody.\n\nThese custom ELISA plates were incubated with 26 human convalescent sera that had been previously characterized as positive for at least one of our target virus taxa using complementary methods, including plaque reduction neutralization test (PRNT) from convalescent serum, IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) from post-acute phase serum, and/or quantitative real-time PCR (qRT-PCR) from blood collected during acute infection. These samples were obtained from public sources including: BEI Resources (5 samples), the World Reference Center for Emerging Viruses and Arboviruses (7 samples), or the United States Centers for Disease Control and Prevention (16 samples).\n\nAfter processing and correcting the raw ELISA data, we found that the well-characterized samples showing a normalized absorbance ratio greater than 1.5 correlated with cases of previously confirmed Zika infection (Table 5–Table 30). Consequently, we compiled the normalized absorbance ratio data and categorized any peptide pool found to have a normalized ratio value greater than 1.5 was classified as a “borderline” result. In order to increase specificity, any sample with at least two wells of the ELISA plate having normalized ratios greater than 1.5 were labeled as putative “positive” for prior infection with the target virus.\n\nGiven the p-values associated with the peptide array results, we decided to especially focus on samples that were positive for ZIKV. As such, instances where excessive signal was detected for all viruses were processed in a way that still identified samples having at least 2x stronger signal for ZIKV peptides than for DENV peptides in the same sample were labeled with a “Z” to differentiate them from other categories.\n\nThe summarized results of the ELISA data revealed a fair amount of concordance with the “gold standard” methods and displayed overall sensitivity and specificity of 61.5% and 50%, respectively (Table 4). Interestingly, these values fluctuated depending on the collection that was analyzed and were affected by small sample size from two of the collections.\n\n\nDiscussion\n\nThe array data reported in this manuscript were used to identify high-scoring peptides that could be used as serodiagnostic reagents in an ELISA format to distinguish between prior infection and seroconversion to a panel of mosquito-borne viruses. Our workflow incorporated both computational and laboratory components to improve identification of regions that were uniquely recognized by virus-specific antibodies to each virus and could therefore be useful as serodiagnostic peptides. Sabalza et al. described a protocol to identify ZIKV specific diagnostic epitopes through peptide microarrays; however, they only used one human serum sample, did not use any bioinformatics analysis, and the identified peptides sequences were not provided38.\n\nThe integration of Gini values calculated by the random forest machine learning algorithm with the BepiPred B-cell epitope prediction algorithm, enabled us to identify the best peptides for each taxon. This approach improved our selected peptides to those that had increased affinity and binding to antibodies33. We purposely chose peptides in both the E and NS1 proteins (E2 protein of CHIKV) to improve our ability to detect epitopes within viral antigens that are known to circulate in the bloodstream11.\n\nWe observed that a few of our selected peptides displayed high reactivity and Gini values, while other selected peptides had lower measured values. We attribute a subset of these unexpected differences to the imposed requirement of being located within a predicted B-cell epitope. Reactivity is an essential measurement for individual samples, while Gini values are useful to rank peptides based on their ability to identify peptides that differentiate one taxon from the others. As such, Gini values are better able to identify linear epitopes that differentiate taxa and that are sufficiently immunodominant across patient populations. We, therefore, are confident in the results from taxa where the Gini values were significantly different between selected peptides when compared to the remaining peptides. By providing the raw data in a publicly-accessible resource, we expect these data to be subject to re-analysis and meta-analysis using alternative methods.\n\nWe also noticed cases where the comparisons of our selected peptides yielded non-significant p-values in various comparisons, especially among dengue viruses. The most likely explanation for this observation is the high degree of cross-reactivity that occurs between linear epitopes derived from these viruses. While other existing serological assays are capable of distinguishing between these highly related taxa, they primarily rely on recognition of conformational epitopes by IgG antibodies circulating in the bloodstream. It is, therefore, possible that linear peptides in the selected proteins may be inadequately suited to differentiate between these taxa. Given the incomplete histories and serology that was performed in a subset of our tested samples, additional work is needed to determine whether incomplete metadata contributed to this finding. Additional laboratory experiments are being performed to calculate the specificity and sensitivity for our sets of peptides in a larger number of human serum samples from various clinical cohorts.\n\nWith these publicly accessible peptide array data, it could also be possible to perform the opposite analysis in a way that would search for regions that were recognized with reduced specificity and could therefore be useful to identify peptides that could indicate past infection by at least one of these viruses. Similarly, these data could be mined to identify linear peptides that could be used as antigens to generate an antibody response to such epitopes towards the development of additional “universal” monoclonal antibodies.\n\nThe ELISA data indicate that this method could be a more resource- and time-efficient approach to PRNT. Although results against alternative characterization methods vary widely, additional criteria have been added to PRNT results to account for the high degree of cross-reactivity between ZIKV and DENV39. The observed sensitivity and specificity values could potentially be improved through additional experimentation and optimization. Screening additional well-characterized samples with our ELISA method could shed additional light into a more accurate gauge of ZIKV seroprevalence and could guide public health decisions.\n\nThese data help to quantify the human humoral response to multiple mosquito-borne viruses and could be useful to identify, map, and/or design native or synthetic antigens that provide increased protection against natural infection by these viruses. Our data could also be relevant to the design of a mosquito-borne virus vaccine. However, care must be taken in designing such experiments to ensure that antibody-dependent enhancement does not increase the risk of adverse events following administration of the vaccine.\n\n\nData availability\n\nFigshare: Peptide Arrays of Three Collections of Human Sera from Patients Infected with Mosquito-Borne Viruses. https://doi.org/10.6084/m9.figshare.c.429860035.\n\nThis project contains the following underlying data:\n\n1 Metadata file for: Information on Peptides Included on Array.\n\n1 Metadata file for: Metadata for Experimental Samples.\n\n151 Data files containing quantitative data for the peptide arrays.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nThe custom script used to parse the data files can be found at GitHub: https://github.com/bpickett/PeptideArray/tree/v0.9.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.351835632.\n\nLicense: GNU General Public License v3.0.",
"appendix": "Acknowledgements\n\nWe gratefully acknowledge the Microarray and NGS Core facility at The Scripps Research Institute, especially Shelby Willis, Ryan McBride, Dr. Phillip Ordoukhanian, and Dr. Steven Head for their excellent technical assistance with preparing and developing the peptide arrays. We also thank Dr. Scott Weaver and Dionna Scharton of the World Reference Center for Emerging Viruses and Arboviruses (NIH AI120942) for their assistance with providing control samples.\n\n\nReferences\n\nD'Ortenzio E, Matheron S, Yazdanpanah Y, et al.: Evidence of Sexual Transmission of Zika Virus. N Engl J Med. 2016; 374(22): 2193–2195. PubMed Abstract | Publisher Full Text\n\nMoreira J, Lamas CC, Siqueira A: Sexual Transmission of Zika Virus: Implications for Clinical Care and Public Health Policy. Clin Infect Dis. 2016; 63(1): 141–2. PubMed Abstract | Publisher Full Text\n\nMcDonald EM, Duggal NK, Brault AC: Pathogenesis and sexual transmission of Spondweni and Zika viruses. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nZambrana JV, Bustos Carrillo F, Burger-Calderon R, et al.: Seroprevalence, risk factor, and spatial analyses of Zika virus infection after the 2016 epidemic in Managua, Nicaragua. Proc Natl Acad Sci U S A. 2018; 115(37): 9294–9299. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalmaseda A, Hammond SN, Tellez Y, et al.: High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua. Trop Med Int Health. 2006; 11(6): 935–42. PubMed Abstract | Publisher Full Text\n\nKatzelnick LC, Gresh L, Halloran ME, et al.: Antibody-dependent enhancement of severe dengue disease in humans. Science. 2017; 358(6365): 929–932. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMattia K, Puffer BA, Williams KL, et al.: Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes. PLoS One. 2011; 6(11): e27252. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMontoya M, Gresh L, Mercado JC, et al.: Symptomatic versus inapparent outcome in repeat dengue virus infections is influenced by the time interval between infections and study year. PLoS Negl Trop Dis. 2013; 7(8): e2357. PubMed Abstract | Publisher Full Text | Free Full Text\n\nbpickett: bpickett/PeptideArray v0.9 (Version v0.9). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3518356\n\nLarsen JE, Lund O, Nielsen M: Improved method for predicting linear B-cell epitopes. Immunome Res. 2006; 2: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamanakumar AV, Thomann P, Candeias JM, et al.: Use of the normalized absorbance ratio as an internal standardization approach to minimize measurement error in enzyme-linked immunosorbent assays for diagnosis of human papillomavirus infection. J Clin Microbiol. 2010; 48(3): 791–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPickett BE, Crowe JE, Harris E, et al.: Peptide Arrays of Three Collections of Human Sera from Patients Infected with Mosquito-Borne Viruses. figshare. Collection. 2019.\n\nJespersen MC, Peters B, Nielsen M, et al.: BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017; 45(W1): W24–W29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMansfield KL, Horton DL, Johnson N, et al.: Flavivirus-induced antibody cross-reactivity. J Gen Virol. 2011; 92(Pt 12): 2821–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSabalza M, Barber CA, Abrams WR, et al.: Zika Virus Specific Diagnostic Epitope Discovery. J Vis Exp. 2017; (130): e56784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRabe IB, Staples JE, Villanueva J, et al.: Interim Guidance for Interpretation of Zika Virus Antibody Test Results. MMWR Morb Mortal Wkly Rep. 2016; 65(21): 543–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "56293",
"date": "14 Nov 2019",
"name": "Salvatore De-Simone",
"expertise": [
"Reviewer Expertise Biochemistry",
"Molecular Biology",
"infection diseases",
"neglected diseases",
"diagnostic",
"immunoassays",
"peptides synthesis",
"microarray of peptides",
"immunochemistry",
"protein structure"
],
"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 focused on an important biomedical problem – How to identify the virus responsible for an infection through serological diagnostic tests when many of the potential pathogens display a high level of sequence similarity? While the approach presented represents an excellent start to achieve their goals, a number of issues need to be addressed. They have correctly focused on short poly-amino acid sequences but need to go further and define the epitome of each virus. As an epitope defines the exact sequence of amino acids bound to the cleft of an immunoglobulin, it is the most precise restriction possible for differentiating similar pathogens. This would allow an in silico evaluation of the potential for cross-reactivity that could be confirmed by immunoassays. Furthermore, a peptide of 15 amino acids could limit reactivity with IgM, which is an important component to defining seroconversion. Another important point is that the panel of patient sera was fairly small. It is also unclear if each patient sample was screened for each of the pathogens in questions or only the suspected infection. It is very common for patients from endemic regions to suffer multiple infections without registering them. A small panel size also makes it difficult to perform Receiver Operating Characteristics analyses, which are absent from the study. In addition, a limited combination of peptides was tested rather than a checkerboard. This is all the more important as peptides often show the lowest sensitivity. Methodologically, another important point to be clarified is regarding the cut off value used in peptide immunoassays or the value used by the software to delimit the signal intensity of a positive test from a negative one.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "5230",
"date": "17 Feb 2020",
"name": "Brett Pickett",
"role": "Author Response",
"response": "We thank the reviewer for sharing their insight about the importance of performing an epitome analysis. We agree that defining the epitome for these cross-reactive viruses would enable more sensitive downstream experimentation with immunoassays. At the time that our study was being performed a very low number of published linear B-cell epitopes existed in the literature. As such, we performed a bioinformatics analysis of existing sequence and structure data to identify linear regions in the three-dimensional protein structure that were both exposed on the protein surface and displayed a number of amino acid substitutions that significantly differed between each of the viral species that we evaluated. We apologize to the reviewer that this prior work was not adequately described in the manuscript. We have included additional text in the last two paragraphs of the Introduction section, as well as the first paragraph of the Methods section, to clarify the purpose and application of our previously published analysis. We believe that the reviewer brings up a very useful point about the limited IgM reactivity of our peptides. It is true that applying these peptides to quantify IgM reactivity could expand the application of the 15-mer peptides as potential serodiagnostics. Although these experiments are important, the majority of the sera we evaluated in this study were collected after the point when IgM levels would produce a high enough signal for detection in these experiments. This is the primary reason that our current study focused on quantifying IgG reactivity. We agree that the number of samples was limited for each of the three sera collections included in this study. The metadata about infection history for each of the samples varied by collection. The sera collections from biorepositories in the United States or Honduras were confirmed during the acute phase with various methods for the target virus. The non-Zika sera from Nicaragua were collected prior to the Zika outbreak, which minimized cross-reactivity. In contrast, the Zika-positive sera from Nicaragua that were included in our study were collected from pediatric patients who were enrolled in an ongoing clinical study and who had no prior infection with Flaviviruses. We have modified the text in the Serum Sources subsection within the Methods section to improve clarity. We appreciate the reviewer pointing out that the ROC analyses were mistakenly left out. We have updated the manuscript with a new display item (Figure 2) that visualizes the results of the ROC analyses for each of the three individual sera collections, as well as for a combination of all collections. The relatively small number of sera was one of the primary reasons why we performed validation of our findings using ELISA. The reviewer’s point describes one of the approaches that are commonly applied to identify important peptides. Our experimental design used a different approach that uses overlapping peptides. Briefly, sets of overlapping peptides, which span a IgG epitope, are included on the array to enable the quantification of antibody binding. Ideally, the signal increases over a set of overlapping peptides until it reaches the specific peptide with optimal antibody binding (and a corresponding peak in signal), followed by a decrease in binding over the remaining peptides. The signal then disappears once the antibody-specific epitope is no longer present on the peptide. Our design takes advantage of this approach at scale to facilitate better identification of peptides containing species-specific epitopes from the peptide array. We thank the reviewer for pointing out this lack of clarity in our methodology. Briefly, normalized ELISA values above 2.5 were categorized as high-confidence, values between 1.5 and 2.5 as medium-confidence, and values below 1.5 as low-confidence. We have added text in the “ELISA Data Processing” subsection of the Methods section to clarify the previously published process."
}
]
},
{
"id": "59099",
"date": "13 Feb 2020",
"name": "Felix F. Loeffler",
"expertise": [
"Reviewer Expertise Microarray technology",
"high-throughput synthesis",
"peptide microarray screening"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 entitled “Peptide arrays of three collections of human sera from patients infected with mosquito-borne viruses” by Pickett et al., the authors report the screening of patient sera using peptide microarrays for antibody interactions. The goal is to identify disease specific biomarkers, since “serological tests are often not reliable for diagnosis after seroconversion and convalescence due to cross-reactivity among flaviviruses”.\nOverall, the approach is sound and has very good potential and the authors did an impressive job in bioinformatic prediction and analysis. Yet, the results show that there is room for technological and methodological improvement.\nI believe that the data presentation can be improved. Thus, I suggest the following minor changes:\nThe title “Peptide arrays of … human sera…” is in my opinion misleading. “Human sera are analyzed/screened on/with peptide arrays” or “Arrays are incubated with human sera”.\n\nIt is somewhat difficult to understand the peptide selection, because there is just a reference to another publication. A brief 2-3 sentences description on the peptide selection process would help.\n\nTo judge the quality of the microarray data, it would be highly beneficial to have some kind of heat map of all array results together (raw and/or normalized median peptide staining intensity per patient). I found it somewhat unconventional to deposit a tsv result file on github. Only providing raw data makes a quick review and validation rather difficult. For an example, see Figure 2 in Weber, L.K., et al. (2017), Eng. Life Sci., 17: 1078-10871. Furthermore, I could not find a link between the raw data files and the corresponding peptide sequence. This needs more explanation and the authors should provide an additional file with the sequences of each position on the array.\n\nIn my opinion, the patient sample presentation has to be improved. The authors state that they only have incomplete patient history (“Given the incomplete histories and serology …additional work is needed to determine whether incomplete metadata contributed to this finding.”). Yet, in the section “Serum sources”, the authors give some ranges on patient age and days after onset of symptoms. In my opinion, a full patient information table (e.g. in supporting information) is essential (even though data might be missing), showing patient age, sex, known previous arbovirus infections (primary vs. secondary infection), days after onset of symptoms, etc. of all patients. From my experience, there can be a large difference in antibody response in patients 10 days vs. 30 days after onset of symptoms, so these patients should be categorized and analyzed as different groups. I believe that patient stratification is one of the keys to more statistically significant data.\nThe following general comments might be helpful to discuss the statistical significance of the results:\nI do not fully agree with this general assumption: “Although reports showing antibodies against other viral proteins are detectable 12, the E and NS1 proteins are the primary targets of the humoral anti-flavivirus immune response in humans 13–15.” Although discussed before (citation 11), this approach ignores for example the highly specific serum biomarker from the NS2B protein in Zika. This approach might miss other similarly important peptide biomarkers. In addition, adding peptides from the nonstructural proteins might help to boost the overall sensitivity of the assay (yet, losing some specificity).\n\nBy choosing consensus sequences, the authors limited the peptide selection, which is valid and – with current array technology – necessary. However, this probably limits the sensitivity of the microarray results and the subsequent ELISA. Depending on the selected protein regions and patient origins (-> virus genotypes), there might be several important genotypic variants, which the authors would have neglected.\nIn addition, the array protocol seems not optimal and there are some inconsistencies in the methods section. Some general remarks, questions, corrections and suggestions regarding the array processing:\nThe raw data states that the scanner Innopsys Innoscan 1100AL and the Mapix software were used. Yet, the in the manuscript, it is stated: “…using a ProScanArray HT (Perkin Elmer) microarray scanner at 488 nm and 600 nm, and images were saved as high-resolution TIF files. Imagene® 6.1 microarray analysis software…”. Please correct. The scanner wavelengths were supposedly 488 nm and 600 nm. In the raw data files, it says 488 nm and 635 nm. Please correct. If possible, please also give the laser powers and gain (PMT) settings for the scans (according to the raw data: wavelengths 635, 488: \"LaserPower=10.0, 5.0\", \"PMTGain=50, 50\").\n\nFor the microarray experiments, the authors claim to have used the Alexa 488 labeled secondary antibody: Invitrogen, cat. #: A-11013, RRID: AB_2534080. This is an (H+L) chain specific antibody, so it will also bind to IgM antibodies (please include this information!)! The use of the Ab labeled with Alexa 488 is a bit odd, since the original data files state that the scanning of the main interaction was performed at 635 nm. Yet, the data of the 488 nm channel is not included in the raw data and, according to the protocol, it seems to me that this dye is only used for controls (see section “Peptide preparation and microarray printing”). Are you sure that you used this specific secondary antibody? If so, I would highly recommend changing to a 635 nm channel compatible secondary antibody, since the 488 nm channel gives much higher background and generally very strong autofluorescence.\n\nIn contrast, for the ELISAs, the authors apparently used a different secondary antibody, which is Fc specific, so it should only bind to IgG. Thus, the microarray data cannot be directly compared to the ELISA data.\n\nThe final steps of the array washing and drying protocol are in my opinion not optimal: “… another two times in deionized water and centrifuged to dry at 200 × g for 5 mins.” The pH of distilled water should be checked. Since deionized water is only weakly buffered (very low salt concentration), deionized water may have a low pH (especially observed in ddH2O). By washing the arrays with it, the lower pH may destroy interactions of weakly (or pH sensitive) binding antibodies. Instead, to remove salt residues from the array surface, you might want to use 1 mM Tris buffer in the future.\n\nIn addition, for future experiments, centrifuging arrays for drying is not optimal, since drying effects may cause artifacts (coffee ring drying effects, etc.). Instead, using a jet of air to quickly remove droplets from the surface causes much less artifacts.\nOverall, the manuscript is well written and the bioinformatics seems sound. Yet, the authors can take some more care in presenting the data and should correct and/or explain the inconsistencies in the methods section (scanner, wavelengths, antibodies).\nFuture analyses should focus on patient stratification and possibly more homogenous samples (different cohorts may be difficult to compare). This may significantly improve statistical outcome.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5262",
"date": "28 Feb 2020",
"name": "Brett Pickett",
"role": "Author Response",
"response": "In this manuscript entitled “Peptide arrays of three collections of human sera from patients infected with mosquito-borne viruses” by Pickett et al., the authors report the screening of patient sera using peptide microarrays for antibody interactions. The goal is to identify disease specific biomarkers, since “serological tests are often not reliable for diagnosis after seroconversion and convalescence due to cross-reactivity among flaviviruses”. Overall, the approach is sound and has very good potential and the authors did an impressive job in bioinformatic prediction and analysis. Yet, the results show that there is room for technological and methodological improvement. I believe that the data presentation can be improved. Thus, I suggest the following minor changes: 1) The title “Peptide arrays of … human sera…” is in my opinion misleading. “Human sera are analyzed/screened on/with peptide arrays” or “Arrays are incubated with human sera”. Response: We thank the reviewer for their input on how to improve the title. We have changed the title to “Peptide arrays incubated with three collections of human sera from patients infected with mosquito-borne viruses”. 2) It is somewhat difficult to understand the peptide selection, because there is just a reference to another publication. A brief 2-3 sentences description on the peptide selection process would help. Response: We agree with the reviewer and have incorporated a more detailed description of the peptide selection process in the last two paragraphs of the Introduction section, as well as the first paragraph of the Methods section in version 2 of the manuscript. We anticipate that these changes sufficiently clarify the peptide selection process that was informed by our previously published analysis. 3) To judge the quality of the microarray data, it would be highly beneficial to have some kind of heat map of all array results together (raw and/or normalized median peptide staining intensity per patient). I found it somewhat unconventional to deposit a tsv result file on github. Only providing raw data makes a quick review and validation rather difficult. For an example, see Figure 2 in Weber, L.K., et al. (2017), Eng. Life Sci., 17: 1078-10871. Furthermore, I could not find a link between the raw data files and the corresponding peptide sequence. This needs more explanation and the authors should provide an additional file with the sequences of each position on the array. Response: We appreciate the reviewer making this useful suggestion. We have generated a heatmap to visually represent the normalized intensity ratios for each peptide predicted to be relevant for ZIKV. This new Figure 2 has been incorporated into the Results section and should adequately address this comment. We apologize that the link between peptide identifiers in the raw data files and the corresponding peptide sequences were not easy to find. The raw data files together with the metadata describing the peptide identifier on the array, the peptide name, and the peptide sequence are publicly available on Figshare (https://doi.org/10.6084/m9.figshare.c.4298600.v2). We have improved the clarity of the file descriptions in the Underlying Data subsection within the Data Availability section of the manuscript. We believe that these adjustments should sufficiently enable readers to link the data files with the peptide sequence. 4) In my opinion, the patient sample presentation has to be improved. The authors state that they only have incomplete patient history (“Given the incomplete histories and serology …additional work is needed to determine whether incomplete metadata contributed to this finding.”). Yet, in the section “Serum sources”, the authors give some ranges on patient age and days after onset of symptoms. In my opinion, a full patient information table (e.g. in supporting information) is essential (even though data might be missing), showing patient age, sex, known previous arbovirus infections (primary vs. secondary infection), days after onset of symptoms, etc. of all patients. From my experience, there can be a large difference in antibody response in patients 10 days vs. 30 days after onset of symptoms, so these patients should be categorized and analyzed as different groups. I believe that patient stratification is one of the keys to more statistically significant data. Response: We agree that providing these additional metadata will be useful in stratifying the data for future analyses and meta-analyses. We have added a new “Patient Metadata” file in the underlying data file collection on FigShare, have updated the Figshare DOI (https://doi.org/10.6084/m9.figshare.c.4298600.v2), and have amended the file description text in the Underlying Data subsection within the Data Availability section of the manuscript. The following general comments might be helpful to discuss the statistical significance of the results: 1. I do not fully agree with this general assumption: “Although reports showing antibodies against other viral proteins are detectable 12, the E and NS1 proteins are the primary targets of the humoral anti-flavivirus immune response in humans 13–15.” Although discussed before (citation 11), this approach ignores for example the highly specific serum biomarker from the NS2B protein in Zika. This approach might miss other similarly important peptide biomarkers. In addition, adding peptides from the nonstructural proteins might help to boost the overall sensitivity of the assay (yet, losing some specificity). Response: We thank the reviewer for pointing out this unclear phrasing. We have corrected the text in the Introduction section to address the usefulness of the nonstructural proteins as potential serological markers. We have also edited the second paragraph of the Discussion section to mention potential future experiments that would incorporate peptides from nonstructural regions. 2. By choosing consensus sequences, the authors limited the peptide selection, which is valid and – with current array technology – necessary. However, this probably limits the sensitivity of the microarray results and the subsequent ELISA. Depending on the selected protein regions and patient origins (-> virus genotypes), there might be several important genotypic variants, which the authors would have neglected. Response: We appreciate the reviewer mentioning the concept that consensus sequence may be affected by strain- or type specific amino acid substitutions. We agree that the usefulness of this approach should be evaluated in future experiments as the capacity and capability of arrays continue to improve. We have added text to inform the readership of this potential weakness of our approach in the fourth paragraph of the Discussion section. In addition, the array protocol seems not optimal and there are some inconsistencies in the methods section. Some general remarks, questions, corrections and suggestions regarding the array processing: 1. The raw data states that the scanner Innopsys Innoscan 1100AL and the Mapix software were used. Yet, the in the manuscript, it is stated: “…using a ProScanArray HT (Perkin Elmer) microarray scanner at 488 nm and 600 nm, and images were saved as high-resolution TIF files. Imagene® 6.1 microarray analysis software…”. Please correct. The scanner wavelengths were supposedly 488 nm and 600 nm. In the raw data files, it says 488 nm and 635 nm. Please correct. If possible, please also give the laser powers and gain (PMT) settings for the scans (according to the raw data: wavelengths 635, 488: \"LaserPower=10.0, 5.0\", \"PMTGain=50, 50\"). Response: We thank the reviewer for finding these inadvertent errors in the text. We have changed the 600 nm wavelength in the manuscript to the correct value of 635 nm, which now matches what is reported in the raw data files. The instrument description in the raw data files is an artifact generated by the scanner software, while the instrument text in the manuscript is correct. We have also included the catalog information for the secondary antibodies, laser power information, and PMT gain in the “High-throughput screening and quantification of the characterized patient sera” subsection of the Methods section. 2. For the microarray experiments, the authors claim to have used the Alexa 488 labeled secondary antibody: Invitrogen, cat. #: A-11013, RRID: AB_2534080. This is an (H+L) chain specific antibody, so it will also bind to IgM antibodies (please include this information!)! The use of the Ab labeled with Alexa 488 is a bit odd, since the original data files state that the scanning of the main interaction was performed at 635 nm. Yet, the data of the 488 nm channel is not included in the raw data and, according to the protocol, it seems to me that this dye is only used for controls (see section “Peptide preparation and microarray printing”). Are you sure that you used this specific secondary antibody? If so, I would highly recommend changing to a 635 nm channel compatible secondary antibody, since the 488 nm channel gives much higher background and generally very strong autofluorescence. Response: We appreciate the reviewer asking us to clarify the text surrounding the secondary antibodies that were used. Indeed, the secondary antibody used to detect serum antibodies bound to viral peptides were in the 635 nm channel, while the 488 nm channel was used solely for the secondary antibodies bound to the control peptides. We have added the correct catalog information for the secondary antibody used to detect reactivity to viral peptides in the “High-throughput screening and quantification of the characterized patient sera” subsection of the Methods section. We have added a new sentence in the third paragraph of the Discussion section to inform the readership that the H+L secondary antibodies that were used could also recognize bound IgM. 3. In contrast, for the ELISAs, the authors apparently used a different secondary antibody, which is Fc specific, so it should only bind to IgG. Thus, the microarray data cannot be directly compared to the ELISA data. Response: The reviewer is correct. The microarray data were generated to narrow down the candidate viral peptides to a much smaller number that would be tractable to test with ELISAs. Changing the secondary antibody between the platforms does affect the ability to directly compare them, and some of the signal loss that was observed could be attributed to that change of reagents. 4. The final steps of the array washing and drying protocol are in my opinion not optimal: “… another two times in deionized water and centrifuged to dry at 200 × g for 5 mins.” The pH of distilled water should be checked. Since deionized water is only weakly buffered (very low salt concentration), deionized water may have a low pH (especially observed in ddH2O). By washing the arrays with it, the lower pH may destroy interactions of weakly (or pH sensitive) binding antibodies. Instead, to remove salt residues from the array surface, you might want to use 1 mM Tris buffer in the future. Response: We appreciate the reviewer sharing this insight and we will take it into account when performing future experiments. 5. In addition, for future experiments, centrifuging arrays for drying is not optimal, since drying effects may cause artifacts (coffee ring drying effects, etc.). Instead, using a jet of air to quickly remove droplets from the surface causes much less artifacts. Response: We thank the reviewer for providing such a useful recommendation to reduce the number of artifacts on the arrays that result from the drying process. We will incorporate this information in future experiments. Overall, the manuscript is well written and the bioinformatics seems sound. Yet, the authors can take some more care in presenting the data and should correct and/or explain the inconsistencies in the methods section (scanner, wavelengths, antibodies). Response: We thank the reviewer for taking the time to review this manuscript so thoroughly and believe that the changes incorporated in the most recent version adequately address the inconsistencies that were found. Future analyses should focus on patient stratification and possibly more homogenous samples (different cohorts may be difficult to compare). This may significantly improve statistical outcome. Response: We agree that stratifying the data in subsequent analyses could improve the findings. We look forward to more samples and data becoming available in the future."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1875
|
https://f1000research.com/articles/8-1027/v1
|
09 Jul 19
|
{
"type": "Research Article",
"title": "CAPE for measuring callous-unemotional traits in disadvantaged families: a cross-sectional validation study",
"authors": [
"Luna C.M. Centifanti",
"Hannah Shaw",
"Katherine J. Atherton",
"Nicholas D. Thomson",
"Susanne MacLellan",
"Paul J. Frick",
"Hannah Shaw",
"Katherine J. Atherton",
"Nicholas D. Thomson",
"Susanne MacLellan",
"Paul J. Frick"
],
"abstract": "Background: Callous-unemotional (CU) traits are important for designating a distinct subgroup of children and adolescents with behaviour problems. As a result, CU traits are now used to form the specifier “with Limited Prosocial Emotions” that is part of the diagnostic criteria for the Conduct Disorder in the Diagnostic and Statistical Manual of Mental Disorders 5th Edition (DSM-5) and International Classification of Diseases 11th Revision (ICD-11). Given this inclusion in major classification systems, it is important to develop and test methods for assessing these traits that can be used in clinical settings. The present study aimed to validate a clinician rating of CU traits, the Clinical Assessment of Prosocial Emotions, Version 1.1 (CAPE 1.1), in a sample of hard-to-reach families referred to a government program designed to prevent the development of behaviour problems in high risk families. Methods: Clinical ratings of children were obtained from 34 families of children ages 3 to 19 (M=12.2; SD=4.3). The ratings on the CAPE 1.1 were based on interviews with both parent and child. Results: Of the sample, 21% (100% male) met the diagnostic cut-off for the specifier according to the CAPE 1.1, and CAPE 1.1 scores were associated with parent ratings of CU traits, psychopathic traits, and externalising behaviours. CAPE 1.1 ratings were also associated with risk for violence obtained from case files. Conclusions: These findings provide preliminary evidence for the validity of the CAPE 1.1 as clinician rated measure of CU traits.",
"keywords": [
"Assessment",
"Callous-Unemotional Traits",
"Conduct Problems",
"Families",
"Personality",
"Externalizing Behavior"
],
"content": "Introduction\n\nCallous-unemotional (CU) traits include behaviours that reflect a lack of caring for others and for doing things well, a lack of guilt and remorse, and a lack of emotional depth in interactions with others1. CU traits across childhood and adolescence have been associated with delinquency, aggression and impairing conduct problems2–4. These traits have proven useful for designating a subgroup of children with serious conduct problems who show distinct emotional, cognitive, and social correlates relative to other youth with behaviour problems5. Further, there is evidence that children with serious behaviour problems who also show elevated levels of CU traits show less positive responses to many interventions used to treat conduct problems6,7. As a result of this evidence for both their clinical and etiological validity, CU traits have recently been included in the specifier “with Limited Prosocial Emotions” (LPE) for the diagnostic criteria for Conduct Disorder in the DSM 5 (Diagnostic and Statistical Manual of Mental Disorders, 5th Edition;8) and for the diagnostic criteria for Oppositional Defiant Disorder and Conduct Disorder in the ICD-11 (International Classification of Diseases version 11;9).\n\nConduct problems are one of the most frequent reasons that youth are referred for mental health treatment10 and they are very costly to the community due to their association with delinquency11. As noted above, those children with serious conduct problems who also have elevated CU traits show less positive responses to many forms of treatment, possibly because of their deficits in emotional responding to the distress in others and abnormalities in their reward and punishment processing12–14. However, certain treatments that are tailored to their unique emotional and cognitive style have shown some limited success in children and adolescents with elevated CU traits7,15,16. Thus, accurate assessment of these traits is critical for treatment planning17.\n\nWith the inclusion in major classification systems used to diagnose childhood behaviour problems and their importance for guiding effective treatment, it is crucial to have measures of CU traits that can be used in high risk samples to make important clinical decisions. This is especially true when children are young, since early interventions tend to be most effective18–20. To date, much of the research on CU traits has relied on informant rating scales that have proven to reliably assess these traits from the age of three years16,21 and in a way that is relatively stable between childhood and adolescence22,23. Further, behaviour ratings provide a very time-efficient method for assessing these traits in research because they are easy to complete and do not require high levels of training to administer and interpret.\n\nHowever, there are significant limitations in the sole reliance on rating scales for making clinical decisions. First, well-established clinical cut-offs are not available for many of these measures to designate when the level of these traits is severe and impairing enough to warrant a diagnosis21,24. Second, as is the case for all forms of psychopathology in children25, ratings of CU traits from different informants often show only modest levels of agreement26. This lack of correspondence across different sources of information makes it a challenge for clinicians to know how best to integrate these discrepant reports when making diagnoses27. Third, a number of biases, both intentional (e.g., deception) and unintentional (e.g., social desirability), can influence ratings of CU traits that need to be considered when making clinical decisions and such sources of bias are often difficult to determine from questionnaires27–29. Fourth, it appears that the optimum assessment of CU traits that detects the construct across all levels of severity requires the use of both positively worded items in which affirmative response denotes more callous and unemotional behaviours (e.g., “do you seem cold and uncaring to others”), as well as negatively worded items in which an affirmative response denotes lower levels of the construct (e.g., “do you feel bad or guilty when you do something wrong”;30). However, negatively worded items can sometimes be difficult to understand and put a strain on a child’s verbal abilities, which are often found to be deficient in children with conduct problems, anyway31. Thus, when making clinical decisions, it is important that items are accurately understood by those providing information. A final issue with existing rating scale measures of CU traits is that they were not developed specifically to assess the criteria for the LPE specifier included in the DSM-5 and ICD-11, which further complicates their use for making the diagnosis21.\n\nTo overcome these limitations of existing measures of CU traits, the Clinical Assessment of Prosocial Emotions, Version 1.1 (CAPE 1.1;32) was developed to assess CU traits in children and adolescents ages 3 to 21 years of age. The CAPE 1.1 was designed specifically to assess the four symptoms of the LPE specifier now included in the DSM 5: lack of guilt/remorse, callousness and lack of empathy, unconcern about performance, and shallow or deficient affect. The CAPE 1.1 uses the structured professional judgement approach to assessment. That is, very explicit descriptions of the four symptoms are provided and experienced clinicians rate the children on each symptom using a three-point scale (0=not at all descriptive; 1=somewhat descriptive; 2=definitely descriptive) based on all sources of information. The manual has descriptions of prototypical behaviour for each of the four items to aid the interviewer in making their ratings. Also, the CAPE 1.1 is explicitly designed to not rely on any single source of information and it provides a semi-structured interview format to aid the clinician in obtaining information from the child, parents, and other informants. These interviews start with stem questions directly related to the items being rated (e.g., “Does ________ seem to care and be concerned about the feelings of others?”). The clinician follows up by asking for examples to ensure that the informant understood the question and by asking other questions (e.g., “Is this how he is most of the time and with most people”) to aid in scoring. The semi-structured format allows the interviewer to probe and ask for as many examples as needed. The final ratings are only made by considering all sources of information and, to conform to DSM-5 criteria, a diagnosis is made when two or more items are scored as being “very descriptive” (the maximum score of 2) of the child.\n\nIn summary, the CAPE 1.1 was designed to overcome many of the limitations of existing measures of CU traits for making clinical diagnoses of the LPE specifier. However, to date, there has been no published data testing the validity of this method. Thus, we investigated the validity of the CAPE 1.1 in a group of socially disadvantaged families that are part of the ‘Troubled Families Scheme’ - implemented by the UK government in 2010. Children who show serious conduct problems tend to come from high-risk backgrounds involving disorganized, unmotivated or disadvantaged families33, and the scheme provides early intervention for these behaviour problems facilitated by local county councils. The scheme targets families in which parents experience unemployment, their children fail to attend school, and where family members are involved (or at risk of being involved) in drug use and/or other criminal activity. The stated aim is to break the cycle of disadvantage that leads to antisocial behaviour across generations. In the current study, we examined the relations among the CAPE 1.1 item scores and criterion-related measures with well-validated questionnaires of CU traits and psychopathic traits in a sample of families in the Troubled Families Scheme. We also validated the CAPE 1.1 with construct-validity measures of offending, and violence from case file records. We examined the relation between the CAPE 1.1 ratings and well-validated questionnaire measures of externalizing (conduct problems) and internalizing behaviours as well as impact of symptoms on daily living. Finally, we examined the validity of the diagnostic cut-off used by the CAPE 1.1 to determine clinical levels of CU traits, as specified in the DSM-5 criteria for the LPE specifier. We decided not to confound the validation measures with CAPE 1.1 ratings, so ratings were solely based on the semi-structured interview.\n\n\nMethod\n\nEthical approval was given by the Psychology Ethics Sub-Committee at University of Durham (approval #12-20). Investigators accompanied a county council caseworker to potential participants’ homes. Inclusion criteria were families enrolled in the “Troubled Families Programme” or the “Family Intervention Project”. Participants were identified by the county council as those families who posed very little risk to investigators’ safety. No other exclusion criteria were necessary. The caseworkers sought permission from families for the investigators to visit to explain the study. At the homes (or in one case, an office), investigators briefed participants and explained that the study was run by Durham University researchers and was separate from the consent process for entering the county council’s programmes. Parents or carers gave written consent to take part for themselves and on behalf of their children. Children were asked for their assent. Families were told all information would be confidential, but exceptions to confidentiality included risk of harm. These families were already in care of the caseworker so the caseworker would have been notified of any risks discovered by investigators and would have notified the police, if necessary. This was never found to be necessary. All investigators held current Disclosure and Barring Service clearance certificates.\n\nParticipants were families who were registered as part of the Troubled Families government scheme from the North East of England. Children’s ages ranged between 3 and 19 years of age (M=12.2; SD=4.3). A total of 34 families took part, which were based on availability and no sample size calculation was performed. Of these families, 24 were enrolled as part of the Troubled Families Scheme, and 10 with a closely aligned (managed by the same team of case workers and with the same interventions offered) Family Intervention Project. These groups did not differ on any of the study measures, so we combined the data. Each family had a target child, whereby the child was identified with the most problematic behaviour or was the focus of the intervention. The questionnaires and interviews that formed the evaluation were completed by the mother of the family and the target child. In one case the mother and father of the child completed the questionnaires and interview together and in another case these were completed by an older sister who was the legal guardian. Other children in the family home were asked to fill out questionnaires (with the investigator reading all items aloud).\n\nThe target child was older on average (M=13.5; SD=3.2; range: 5–18 years) than the non-target children in the same families, typically biological siblings (M=10.9; SD=4.7; range 3–22 years). With regards to gender, 24 (69%) of the 35 target children (one family had two target children) were male, while 25 out of 50 (50%) of the non-targets were male. The difference between the target groups on gender (Χ2= 2.91, p = .09) was not significant.\n\nOne of the target children was selected at random from the family that had two target children.\n\nFamilies already targeted by the Troubled Families scheme were contacted by the Stockton Community Safety Team to be invited to take part in this study. They were told that participation was voluntary and that their decision would not affect involvement in the ‘Troubled Families’ scheme. Participants did not receive incentives for participating. We used the judgement of the case workers regarding safety to enter family homes, and we were always accompanied by one case worker for the hour in which we conducted assessments. Only 6 families were deemed unsuitable or declined, within the time frame in which we conducted the assessments (from December 2013 to September 2014). Investigators collected data at participants’ homes (except for one case, which was completed in a private room at the county council building). Parents gave written consent to take part for themselves and on behalf of their children; children were verbally asked for assent.\n\nLinks to all measures used in this study are provided as extended data\n\nClinical Assessment of Prosocial Emotions, Version 1.1 (CAPE 1.1;32). The CAPE 1.1 was scored in the current study based on semi-structured interviews conducted with the target child and parent in all but three cases. For two children below the age of 7 and for a child with a diagnosis of Autism Spectrum Disorder, only the parent interview was conducted. While the CAPE 1.1 is designed to use all sources of information available to the clinician, including semi-structured interviews, rating scales, and file review, the scoring in the current study was based solely on the interviews, so that rating scales and file information could be used to test the validity of the CAPE 1.1 scores. Clinicians rated the four items of CAPE 1.1 using the 3-points described previously. The CAPE 1.1 was carried out by the first and fourth authors (LCMC and NDT), who despite not being registered clinicians had a sufficient level of qualification and training to carry out the assessments. The first author is trained in psychometric assessments within clinical settings and has completed a PhD that involved carrying out psychoeducational and mental health assessments within a community clinic, special education provision, and juvenile justice services under the supervision of a licensed clinical psychologist in the USA. The fourth author is a trained psychotherapeutic counsellor accredited and registered with UK Council for Psychotherapists as a therapist and counsellor, with a clinical psychology master’s degree and experience working as a psychotherapist in a prison in the USA as well as subsequent independent clinic work in the northeast of England. We used scores from the CAPE 1.1 in two ways. First, we used the number of symptoms rated categorically as being “very descriptive”: the number of items rated “2”. Thus, the range was 0 to 4. Second, we examined the prevalence and validity of the diagnostic threshold used by the CAPE 1.1 to approximate the Limited Prosocial Emotions specifier, as defined in the DSM 58, which defines those with the diagnosis as persons with 2 or more symptoms rated as 2.\n\nChild Problematic Traits Inventory (CPTI;34,35). The CPTI is a 28-item measure that was originally developed for teachers to report on psychopathic-like traits for children, but it has been used to obtain parent-reports in one previous investigation27. Thus, we used the parent reports on the total score (α = .93) as well as the three sub-scales measuring CU traits (α = .91), grandiosity/deception (α = .90), and impulsivity/need for stimulation (α = .85), since we were interested in the relations with psychopathic traits in general.\n\nStrengths and Difficulties Questionnaire (SDQ;36). The SDQ is a 25-item scale that assesses five domains of adjustment including Hyperactivity, Conduct Problems, Emotional Symptoms, Peer Problems, and Prosocial Behavior. Generally, the SDQ was found to be a reliable and valid measure of conduct problems and has been widely used in both community and clinical samples of children and adolescents37. A three-subscale division has been recommended by Goodman et al.38 consisting of externalizing behaviour, internalizing behaviour and the prosocial scale. Externalizing behaviour was assessed by combining the Hyperactivity and Conduct Problems sub-scales, on both a parent (α = 0.89) and child (α = 0.75) version. Internalizing behaviour was also assessed using the child (α = 0.48) and parent (α = 0.76) versions, combining Emotional Symptoms and Peer Problems. The prosocial scale was utilized as part of the University of New South Wales (UNSW) CU Traits measure (see below). In addition, part of the extended version of the SDQ, the impact supplement was used to assess further chronicity, social impairment, distress and burden to others39. This is a scale, based on parent-reports, that sums items about impact on free time and leisure, home activities, and school activities, as well as distress caused by symptoms.\n\nUNSW CU Traits40. The UNSW measure of CU traits has been created from items on the prosocial (5 items) and conduct problems (1 item) sub-scales of the SDQ. Additionally, three items from the Antisocial Process Screening Device (APSD;41) are included in this scale. This measure was created based on a factor analytic assessment of the SDQ and APSD and has since been extensively validated42,43. The 9-item measure was collected using parent (α = 0.87) and child (α = 0.78) reports.\n\nCase file records. A dichotomous measure (1=present, 0=not present) was created from the risk assessment for violence that caseworkers assessed as part of their work with the families. We also created dichotomous variables of the target child’s involvement (1=yes, 0=no) with Young offending services (YOS) as a measure of delinquent behaviour since this service (from the UK’s National Health Service) is concerned with treating and rehabilitating juveniles who have engaged in delinquency.\n\nJASP 0.9.1.044 was used for t-tests, descriptive statistics, and chi-square analyses. The package ggcorrplot 0.1.245 in R 1.1.45346 software was used for correlation analyses. Seven of the target youths refused to participate in filling out the questionnaires, so correlations with the CAPE reflect this when using child-report, but parent-report was available. For one of the target children, researcher error meant that the parent only completed the CPTI but not the SDQ.\n\n\nResults\n\nWe first tested whether target children for the intervention scored higher on emotional and behaviour problems, as well as CU and psychopathic traits. The mean scores and standard deviations are shown in Figure 1 (see underlying data47). Welch t-tests are reported because of violations of normality assumptions. Children targeted for the intervention were higher than their siblings (non-target children) in parent-reported psychopathic traits on the CPTI (t(62.20)= -3.14, p = 0.003, d = -0.72), and this included all subscales: callous/unemotional (t(68.25)= -2.25, p = 0.027, d = -0.51), grandiosity/deception (t(56.50)= -2.96, p = 0.005, d = -0.69), and impulsivity/need for stimulation subscale (t(70.08)= -2.64, p = 0.010, d = -0.60). Target children were higher than non-target children in UNSW CU traits according to both parents and children themselves (t(65.50)= -2.38, p = 0.020, d = -0.54; t(38.83)= -2.61, p = 0.013, d = -0.80) as well as parent-reported externalizing behaviour (t(68.30)= -2.33, p = 0.023, d = -0.53) and they showed more of an adverse impact on their daily living activities from their symptoms based on the SDQ (t(53.19)= -4.11, p = < 0.001, d = -0.96). Thus, target children were similar to their non-target siblings in symptoms of internalizing behaviour problems, regardless of reporter, but were higher on psychopathic traits, CU traits and the impact of their problems on their activities of daily living.\n\n(Note: CPTI= Child Problematic Traits Inventory, GD= Grandiosity/deception, CU= Callous/unemotional, INS=Impulsivity/need-for-stimulation, UNSW= University of New South Wales, CR= Child-report, PR= Parent-report).\n\nFigure 2 shows the percentage of target children that scored at each level of severity for each symptom on the CAPE 1.1. The most frequent rating given to children was ‘0’ for all symptoms, ranging from 41% to 59% across the four symptoms. The least frequent rating was the maximum score of “2”, which ranged from 15% to 29% across symptoms. Of the 34 target children, seven children (21%; all males) met diagnostic criteria for the LPE specifier (two or more items were rated at a maximum value of ‘2’). Thus, the majority of the sample did not meet the threshold for the diagnosis, and most did not have symptoms that reached a clinical range.\n\nTo determine whether all items equally discriminated those meeting diagnostic criteria on the CAPE, we examined the frequency of symptom scores within those who did and not meet the threshold for the LPE specifier. The results are provided in Figure 3. For ‘lack of remorse or guilt’, ‘callous-lack of empathy’ and ‘shallow or deficient affect,’ the majority of the seven children who met criteria for the LPE specifier were rated a ‘2’, meaning their behaviour was believed to be very descriptive on these three symptoms. No children meeting the criteria for LPE specifier scored ‘0’ for ‘lack of remorse or guilt’ or ‘callous-lack of empathy’ and only one of these children scored ‘0’ for ‘shallow or deficient affect.’ In contrast, the ratings for ‘unconcerned about performance’ did not seem to differ between children who met the criteria for the LPE specifier and those who did not.\n\nFigure 4 notes the results of Spearman’s correlations examining the associations of the validation measures (questionnaires and case file records) with CAPE 1.1 ratings (number of symptoms rated 2). An ‘x’ placed across the correlation in the figure denotes that the result is non-significant. Spearman’s rho was used because of the non-parametric nature of many of the measures utilized. CAPE 1.1 scores were significantly correlated with CU traits (when measured by parent report only), psychopathic traits, ratings of violence from their case files, and the negative impact of their mental health symptoms on their daily living. There were no associations found between the CAPE 1.1 and externalizing/internalizing behaviour regardless of reporter, although the relation with externalizing behaviour (as reported by the parent) was moderate.\n\n(Note: CPTI= Child Problematic Traits Inventory, GD= Grandiosity/deception, CU= Callous/unemotional, INS=Impulsivity/need-for-stimulation, UNSW= University of New South Wales, Child= Child-report, Parent= Parent-report, CAPE= Clinical Assessment of Prosocial Emotions).\n\nThe final test of validity focused on whether the children who met criteria for the LPE specifier (n =7) would be different from those who did not (n = 27) on the various measures of problem behaviour and questionnaire measures of psychopathic traits. The mean scores and standard deviations for the parent and child report measures across the two groups are shown in Figure 5. Levene’s test of equality of variance was nonsignificant except for child reported internalizing behaviour, so we used Student t-tests except in that case. Those children diagnosed with the LPE specifier were found to have higher CU traits according to the UNSW parent report (t(31)= -3.02, p = 0.005, d = -1.29) and higher psychopathic traits (t(32)= -2.32, p = 0.027, d = -0.99) according to the CPTI total – specifically, they were higher on the grandiosity/deception subscale (t(32)= -2.84, p = 0.008, d = -1.20) and the CU subscale (t(32)= -2.32, p = 0.027, d = -0.99). However, the two groups did not differ on the impulsivity/need for stimulation subscale of the CPTI (t(32)= -0.84, p = 0.408, d = -0.36) or any of the other measures. With regard to case file records, children meeting the criteria for the LPE specifier did not differ, from those not meeting criteria, on risk for violence or for contact with young offending services (X2 (1, N = 32) = 1.52, p = .217; X2 (1, N = 32) = 0.96, p = 0.327, respectively). Of note, no girls met criteria for the LPE specifier.\n\n\nDiscussion\n\nIn a sample of difficult-to-engage families, we showed that clinician ratings of CU traits using the CAPE 1.1 were associated with parent ratings of CU traits, psychopathic traits, and externalising behaviours. CAPE 1.1 ratings were also associated with risk for violence obtained from case files. These findings provide preliminary evidence for the validity of the CAPE 1.1 as a measure of CU traits, assessed in a way that is consistent with the Limited Prosocial Emotions (LPE) specifier for the diagnosis of Conduct Disorder included in the DSM-5 and ICD-11.\n\nAs the CAPE 1.1 and UNSW CU traits measure are both derived from the ASPD and are therefore based on the same historical items, it is possible that they may use similarly worded items. However, as the CPTI was created differently, significant association between the CAPE 1.1 scores and the CU subscale of the CPTI suggests that findings do not solely rely on the validity between similarly worded items.\n\nUsing the diagnostic cut-off specified by the CAPE 1.1, we found that 21% of the target children met criteria for the LPE specifier, which is commensurate with estimates from prior research using detained48,49, clinic-referred50, and high-risk51 samples. When using this cut-off, those who scored above the diagnostic threshold were higher on parent ratings of CU traits and also ratings of grandiosity and deceptiveness, which are part of the interpersonal psychopathic traits. However, this group did not differ on the other measures of externalizing behaviour or risk for violence. There are two possible reasons for this finding. First, the cut-off led to only 7 children in the sample meeting the diagnostic criteria, leading to very low power for detecting group differences. Second, the use of a high-risk sample meant that even those who were not elevated on CU traits likely showed significant problems in adjustment.\n\nThe findings provide preliminary evidence for the diagnostic cut-off specified in the CAPE 1.1 manual as an appropriate measure to designate when the level of these traits warrants a diagnosis. CAPE 1.1 ratings using the sum of items scored at the maximum of “2” were significantly correlated with ratings of violence. This signifies that the prototypical presentation of low empathy, lack of guilt, lack of concern and shallow affect was associated with greater problems in adjustment across reporter-based measures but also case file records. Additionally, the greater number of areas where children showed this prototypicality were even more related to maladjustment. Further research with larger samples and with more diversity of problem behaviours is required to determine if the diagnostic cut-off is useful for this tool.\n\nImportantly, the CAPE 1.1 provides a structured method for making clinical decisions, making it potentially useful for clinical settings that require more in-depth assessments than reliance simply on scores from rating scales. Clinical uses will become increasingly common now that CU traits are included in the diagnostic classification systems used globally. Further, as interventions are developed and tested to specifically target the needs of children and adolescents with elevated CU traits52, their success will rely on adequate assessment, especially in samples who experience social disadvantage, behaviour problems, poor school attendance, and who show behaviours that are generally difficult to access. Further, in such samples, clinical judgement will be important to ensure that reporters understand the questions and are able to provide information in a way that is appropriate for their cultural and educational background. Specifically, the CAPE 1.1 requires clinicians to gain examples from the informants in their own words, to ensure that the questions are understood and answered in the way that is intended.\n\nGiven that the CAPE 1.1 only leads to the rating of four items, it was somewhat surprising that they still formed a relatively internally consistent scale. This finding suggests that scores from the CAPE 1.1 can be used as a continuous measure of CU traits as well. Further, three of the four items differentiated those who scored above the diagnostic criterion from those who did not. Specifically, the frequency at which the item “unconcerned about performance” was rated as being at the symptom level (i.e., a score of 2 or “very descriptive”) was similar for children who did and did not meet the diagnostic criteria for LPE. The small sample leading to a limited number of children meeting criteria for the specifier, again suggests that this finding should be replicated in other samples. However, it does point to the need to evaluate the relative utility of the symptoms used to define the LPE specifier50.\n\nThese results need to be interpreted in light of several limitations. First, as noted previously, the sample size led to a very small number of children meeting the criteria for the LPE specifier and thus, there was limited power of the tests comparing those meeting and those not meeting the diagnostic threshold for the LPE specifier. The small sample also prevented us from testing potential moderators of the validity of CAPE 1.1. Of particular note, we could not test potential differences that might have been found in the validity of the CAPE 1.1 across age or sex of the child. Second, we only used parent and child reports from semi-structured interviews to score the CAPE 1.1; we did not have access to teachers as potential informants. Also, because we wanted to use behaviour ratings and case files to test the validity of the CAPE 1.1 scores, clinicians were not allowed to use this information in their ratings. Thus, clinicians were not able to use “all sources of available information”, as recommended by the CAPE 1.1 manual32. Third, although the researchers carrying out the CAPE 1.1 had a sufficient level of training to do so (as previously outlined), they would not meet the criteria to make clinical decisions from its use relative to what is recommended in the manual. However, as the CAPE 1.1 was carried out solely for research purposes, rather than diagnostic purposes, that level of training was not necessary. Fourth, because of the training required and the method of obtaining information that relies on collecting multiple sources of information, the CAPE 1.1 is a much more time consuming and expensive method for assessing CU traits when compared to behaviour rating scales. While we have argued that this could be beneficial for many clinical uses, it will be important for future research to test whether the scores from the CAPE 1.1 provide important information over and above that provided by rating scales that would justify the cost.\n\nIn sum, the CAPE 1.1 shows promise as a method for assessing CU traits in a way that is a) consistent with the diagnostic criteria for the LPE specifier and b) useful for making complex clinical decisions. Further, this promise was demonstrated in a sample of hard-to-reach families for whom clinical decisions may be difficult to make through other means. As a result, the CAPE 1.1 could provide a method for making important clinical decisions for children and adolescents who are at risk for a particularly severe and chronic pattern of conduct problems, for whom careful treatment planning is essential.\n\n\nData availability\n\nFigshare: Centifanti TF target-child dataset for limited prosocial emotions using CAPE. https://doi.org/10.6084/m9.figshare.830029747\n\nThis project contains the following underlying data:\n\nTF open data.csv (CAPE data for target children for whom the CAPE was completed. Sibling and family level data were removed for privacy)\n\nvariable names.txt (Codebook for underlying data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nClinical Assessment of Prosocial Emotions, Version 1.1 (CAPE 1.1;32): https://sites01.lsu.edu/faculty/pfricklab/cape/\n\nChild Problematic Traits Inventory (CPTI;34,35): https://www.oru.se/english/research/research-environments/hs/caps/cpti/\n\nStrengths and Difficulties Questionnaire (SDQ;36): http://www.sdqinfo.com/\n\nUniversity of New South Wales (UNSW) Callous-Unemotional (CU) Traits40: https://psycnet.apa.org/fulltext/2005-06517-003.pdf",
"appendix": "Grant information\n\nThis study was not externally funded except through consultancy with Stockton-on-Tees County Council given to LCMC.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nFrick PJ, Ray JV: Evaluating Callous-Unemotional Traits as a Personality Construct. J Pers. 2015; 83(6): 710–722. PubMed Abstract | Publisher Full Text\n\nFrick PJ, Robertson EL, Clark JE: Callous–unemotional traits. Developmental Pathways to Disruptive, Impulse-Control and Conduct Disorders. Elsevier. 2018; 139–160.\n\nFontaine NM, Rijsdijk FV, McCrory EJ, et al.: Etiology of different developmental trajectories of callous-unemotional traits. J Am Acad Child Adolesc Psychiatry. 2010; 49(7): 656–664. PubMed Abstract | Publisher Full Text\n\nFrick PJ, White SF: Research review: the importance of callous-unemotional traits for developmental models of aggressive and antisocial behavior. 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Publisher Full Text\n\nKimonis ER, Fanti KA, Frick PJ, et al.: Using self-reported callous-unemotional traits to cross-nationally assess the DSM-5 'With Limited Prosocial Emotions' specifier. J Child Psychol Psychiatry. 2015; 56(11): 1249–1261. PubMed Abstract | Publisher Full Text\n\nFrick PJ, Ray JV, Thornton LC, et al.: Annual research review: A developmental psychopathology approach to understanding callous-unemotional traits in children and adolescents with serious conduct problems. J Child Psychol Psychiatry. 2014; 55(6): 532–548. PubMed Abstract | Publisher Full Text\n\nMuñoz LC, Frick PJ: The reliability, stability, and predictive utility of the self-report version of the Antisocial Process Screening Device. Scand J Psychol. 2007; 48(4): 299–312. PubMed Abstract | Publisher Full Text\n\nDocherty M, Boxer P, Huesmann LR, et al.: Assessing Callous-Unemotional Traits in Adolescents: Determining Cutoff Scores for the Inventory of Callous and Unemotional Traits. J Clin Psychol. 2017; 73(3): 257–278. PubMed Abstract | Publisher Full Text\n\nDe Los Reyes A, Kazdin AE: Informant discrepancies in the assessment of childhood psychopathology: a critical review, theoretical framework, and recommendations for further study. Psychol Bull. 2005; 131(4): 483–509. PubMed Abstract | Publisher Full Text\n\nRoose A, Bijttebier P, Decoene S, et al.: Assessing the affective features of psychopathy in adolescence: a further validation of the inventory of callous and unemotional traits. Assessment. 2010; 17(1): 44–57. PubMed Abstract | Publisher Full Text\n\nSomma A, Andershed H, Borroni S, et al.: The validity of the child problematic trait inventory in 6–12 year old italian children: further support and issues of consistency across different sources of information and different samples. J Psychopathol Behav Assess. 2016; 38(3): 350–372. Publisher Full Text\n\nCoid J, Yang M, Ullrich S, et al.: Psychopathy among prisoners in England and Wales. Int J Law Psychiatry. 2009; 32(3): 134–141. PubMed Abstract | Publisher Full Text\n\nPerenc L, Radochoński M: Prevalence of psychopathic traits in a large sample of Polish adolescents from rural and urban areas. Ann Agric Environ Med. 2016; 23(2): 368–372. PubMed Abstract | Publisher Full Text\n\nRay JV, Thornton LC, Frick PJ, et al.: Impulse Control and Callous-Unemotional Traits Distinguish Patterns of Delinquency and Substance Use in Justice Involved Adolescents: Examining the Moderating Role of Neighborhood Context. J Abnorm Child Psychol. 2016; 44(3): 599–611. PubMed Abstract | Publisher Full Text\n\nSmith AK, Stasi SM, Rhee SH, et al.: The Role of Attention-Deficit/hyperactivity Disorder in the Association between Verbal Ability and Conduct Disorder. Front Psychiatry. 2011; 2: 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrick PJ: CLINICAL ASSESSMENT OF PROSOCIAL EMOTIONS: VERSION 1.1 (CAPE 1.1). University of New Orleans. 2013; 1–27. Reference Source\n\nMeltzer H, Gatward R, Goodman R, et al.: The mental health of children and adolescents in Great Britain. dawba.com; 2000. Reference Source\n\nColins OF, Andershed H, Frogner L, et al.: A new measure to assess psychopathic personality in children: the child problematic traits inventory. J Psychopathol Behav Assess. 2014; 36(1): 4–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nColins OF, Fanti K, Larsson H, et al.: Psychopathic Traits in Early Childhood: Further Validation of the Child Problematic Traits Inventory. Assessment. 2017; 24(5): 602–614. PubMed Abstract | Publisher Full Text\n\nGoodman R: The Strengths and Difficulties Questionnaire: A Research Note. J Child Psychol Psychiatry, 1997; 38(5): 581–586. PubMed Abstract | Publisher Full Text\n\nViding E, Frick PJ, Plomin R: Aetiology of the relationship between callous-unemotional traits and conduct problems in childhood. Br J Psychiatry Suppl. 2007; 49: s33–8. PubMed Abstract | Publisher Full Text\n\nGoodman A, Lamping DL, Ploubidis GB: When to use broader internalising and externalising subscales instead of the hypothesised five subscales on the Strengths and Difficulties Questionnaire (SDQ): data from British parents, teachers and children. J Abnorm Child Psychol. 2010; 38(8): 1179–1191. PubMed Abstract | Publisher Full Text\n\nGoodman R: The extended version of the Strengths and Difficulties Questionnaire as a guide to child psychiatric caseness and consequent burden. J Child Psychol Psychiatry. 1999; 40(5): 791–799. PubMed Abstract | Publisher Full Text\n\nDadds MR, Fraser J, Frost A, et al.: Disentangling the underlying dimensions of psychopathy and conduct problems in childhood: a community study. J Consult Clin Psychol. 2005; 73(3): 400–410. PubMed Abstract | Publisher Full Text\n\nFrick PJ, Hare RD: The antisocial process screening device. Toronto: Multi-Health Systems; 2001.\n\nDadds MR, Allen JL, McGregor K, et al.: Callous-unemotional traits in children and mechanisms of impaired eye contact during expressions of love: a treatment target? J Child Psychol Psychiatry. 2014; 55(7): 771–780. PubMed Abstract | Publisher Full Text\n\nMoul C, Dobson-Stone C, Brennan J, et al.: An exploration of the serotonin system in antisocial boys with high levels of callous-unemotional traits. PLoS One. 2013; 8(2): e56619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeam J: JASP (version 0.9)[computer software].\n\nKassambara A: ggcorrplot: Visualization of a Correlation Matrix using’ggplot2. 2016. Reference Source\n\nTeam RC: R: A language and environment for statistical computing. 2013.\n\nCentifanti L: Centifanti TF target-child dataset for limited prosocial emotions using CAPE. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8300297.v1\n\nColins OF, Andershed H: The DSM-5 with limited prosocial emotions specifier for conduct disorder among detained girls. Law Hum Behav. 2015; 39(2): 198–207. PubMed Abstract | Publisher Full Text\n\nVan Damme L, Colins OF, Vanderplasschen W: The Limited Prosocial Emotions Specifier for Conduct Disorder Among Detained Girls: A Multi-Informant Approach. Crim Justice Behav. 2016; 43(6): 778–792. Publisher Full Text\n\nKahn RE, Frick PJ, Youngstrom E, et al.: The effects of including a callous-unemotional specifier for the diagnosis of conduct disorder. J Child Psychol Psychiatry. 2012; 53(3): 271–282. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPardini D, Stepp S, Hipwell A, et al.: The clinical utility of the proposed DSM-5 callous-unemotional subtype of conduct disorder in young girls. J Am Acad Child Adolesc Psychiatry. 2012; 51(1): 62–73.e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKimonis ER, Fleming G, Briggs N, et al.: Parent-Child Interaction Therapy Adapted for Preschoolers with Callous-Unemotional Traits: An Open Trial Pilot Study. J Clin Child Adolesc Psychol. 2019; 48(sup1): S347–S361. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "54368",
"date": "01 Oct 2019",
"name": "Stuart F. White",
"expertise": [
"Reviewer Expertise Developmental Psychopathology & Cognitive Neuroscience."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current manuscript investigates the CAPE assessment in a very difficult to access population. While there is some indication of the validity and utility of the CAPE from these data, the impact of the findings is diminished by the small sample size.\n\nIntroduction\nPlease reference both the “Troubled Families Programme,” and the “Family Intervention Project” in the introduction, or just say “drawn from government intervention programs” or similar. It was slightly confusing tracking back to the introduction from the methods when I expected only one program and two were described.\n\nPlease discuss why the non-target youth were assessed in this study in the introduction.\nMethods/Results\nThe authors write, “no sample size calculation was performed.” I suspect that the authors mean that no power analysis was conducted to inform sample size. Please be clear.\n\nThe level of detail regarding the clinical training of the interviewers seems a bit excessive. Something along the lines of “the interviewers had sufficient clinical training and research experience to conduct the interviews, though were not registered clinicians” should suffice. That the interviews were not conducted by the typical clinician is a weakness that should be discussed later in the paper.\n\nThe comparison between target and non-target youth comes a bit from no-where. This analysis is not well set-up in the introduction. Please make it clear why this analysis is conducted in the introduction.\n\nIn Figures 1 & 5, please report standard error bars, as opposed to standard deviation.\n\nThe sentence “There were no associations found between the CAPE 1.1 and externalizing/internalizing behavior regardless of reporter, although the relation with externalizing behaviour (as reported by the parent) was moderate” is unclear. If there was no association, how can the relationship be moderate?\n\nFigure 4 is very difficult to read - it is a jumble. Perhaps indicate via color-code specific ranges of correlation/significant correlations. The specific numbers and the crossing out of those numbers is less than ideal.\nDiscussion\nThe non-target children are not mentioned in the discussion. They either need to be incorporated more fully into the paper or removed from the paper.\n\nI think that the authors need to spend more time in the discussion making the case that these data are able to help clinicians evaluate the CAPE. The authors did an admirable job of accessing a difficult population, but the reality is that the numbers are so low, it’s hard to interpret the data. This is especially the case since the CAPE wasn’t used entirely in a manner consistent with recommended clinical use. Why would a clinician, after reading this paper, make a decision one way or the other about using the CAPE in clinical practice? The discussion needs to take this criticism on directly and forcefully.\n\nMinor points:\nThe following sentence is clunky: “However, negatively worded items can sometimes be difficult to understand and put a strain on a child’s verbal abilities, which are often found to be deficient in children with conduct problems, anyway.” Perhaps revise?\n\n“No other exclusion criteria were necessary” is an odd way to write this. Please be clear that no other exclusion criteria were employed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5256",
"date": "27 Feb 2020",
"name": "Luna Centifanti",
"role": "Author Response",
"response": "Thank-you for the opportunity to revise and resubmit our paper to F1000Research. We are very glad to hear that there was interest in our manuscript and an appreciation for the contribution made with regards to providing support for the utility and validity of the CAPE. We would like to thank the three reviewers for their insightful and constructive comments on the first submission. Below we have responded to their comments and hope to have addressed the concerns adequately. Reviewer 1 Introduction (a) Please reference both the “Troubled Families Programme,” and the “Family Intervention Project” in the introduction, or just say “drawn from government intervention programs” or similar. It was slightly confusing tracking back to the introduction from the methods when I expected only one program and two were described. Thank-you for this comment, we agree with this point and have changed the introduction and the method section of the paper to make this clearer. (a) Please discuss why the non-target youth were assessed in this study in the introduction. In light of the number of comments made surrounding the inclusion of the non-target youth, we have made the decision to remove this part of the assessment. As the paper is about validating the CAPE, we want to ensure this is where the focus remains. Methods/Results (b) The authors write, “no sample size calculation was performed.” I suspect that the authors mean that no power analysis was conducted to inform sample size. Please be clear. This is what was meant, so we have changed “no sample size calculation was performed” to “no power analysis was conducted to inform sample size,” as suggested. (b) The level of detail regarding the clinical training of the interviewers seems a bit excessive. Something along the lines of “the interviewers had sufficient clinical training and research experience to conduct the interviews, though were not registered clinicians” should suffice. That the interviews were not conducted by the typical clinician is a weakness that should be discussed later in the paper. We included the specific qualifications of the interviewers for transparency but agree that this level of detail for the purposes of the paper is excessive. Therefore, we have cut down the information provided. The CAPE 1.1 was carried out by the first and fourth authors (LCMC and NDT), who despite not being registered clinicians had a sufficient level of qualification, clinical training and research experience to carry out the assessments. (b) The comparison between target and non-target youth comes a bit from no-where. This analysis is not well set-up in the introduction. Please make it clear why this analysis is conducted in the introduction. We agree with this point and have removed this part of the assessment. This is outlined in response to [Reviewer 1’s] comment 2 (a). (b) In Figures 1 & 5, please report standard error bars, as opposed to standard deviation. Figure 1 has been removed from the paper as we have made the decision to remove this part of the assessment (see response to [Reviewer 1’s] comment 2a). Figure 5 is now referred to as Figure 4 and standard deviations have been changed to standard error bars. (b) The sentence “There were no associations found between the CAPE 1.1 and externalizing/internalizing behavior regardless of reporter, although the relation with externalizing behaviour (as reported by the parent) was moderate” is unclear. If there was no association, how can the relationship be moderate? Thank-you for noticing this, this was in error and we have corrected it. CAPE 1.1 scores were significantly correlated with CU traits (when measured by parent report only), externalising behaviour (when measured by parent report only), psychopathic traits, ratings of violence from their case files, and the negative impact of their mental health symptoms on their daily living. There were no associations found between the CAPE 1.1 and internalizing behaviour regardless of reporter. (b) Figure 4 is very difficult to read - it is a jumble. Perhaps indicate via color-code specific ranges of correlation/significant correlations. The specific numbers and the crossing out of those numbers is less than ideal. To make the information easier to read, we have now presented this in a correlation table. Discussion (C) The non-target children are not mentioned in the discussion. They either need to be incorporated more fully into the paper or removed from the paper. We agree with this point and have removed this part of the assessment. This is outlined in response to [Reviewer 1’s] comment 2 (a). (C) I think that the authors need to spend more time in the discussion making the case that these data are able to help clinicians evaluate the CAPE. The authors did an admirable job of accessing a difficult population, but the reality is that the numbers are so low, it’s hard to interpret the data. This is especially the case since the CAPE wasn’t used entirely in a manner consistent with recommended clinical use. Why would a clinician, after reading this paper, make a decision one way or the other about using the CAPE in clinical practice? The discussion needs to take this criticism on directly and forcefully. Thank-you for this comment. We have added a paragraph to the discussion to address why we think the CAPE would be useful in clinical practice. Minor points: (D) The following sentence is clunky: “However, negatively worded items can sometimes be difficult to understand and put a strain on a child’s verbal abilities, which are often found to be deficient in children with conduct problems, anyway.” Perhaps revise? We have taken some words out of this sentence in the hope that it is now easier to read. However, negatively worded items can be difficult to understand and put a strain on a child’s verbal abilities, which have been found to be deficient in children with conduct problems. (D) “No other exclusion criteria were necessary” is an odd way to write this. Please be clear that no other exclusion criteria were employed. Thank-you for noticing this. We have changed ‘no other exclusion criteria were necessary’ to ‘no other exclusion criteria were employed,’ as suggested."
}
]
},
{
"id": "56051",
"date": "07 Nov 2019",
"name": "Giorgos Georgiou",
"expertise": [
"Reviewer Expertise Callous Unemotional traits",
"empathy",
"physiological measurements",
"conduct problems"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting manuscript investigates the CAPE 1.1 assessment in a difficult to access population range from 3 - 19 years old. While there is some indication regarding utility and validity of the CAPE 1.1, the impact of the findings is diminished by the small sample size and some minor issues.\nIntroduction:\n\n1. Authors provide very detailed information regarding CAPE 1.1 and also the aim of the study. However, since authors investigated the difference between non-target with target children on several measures, something that is not mentioned in the introduction, they should revised aim section by adding this information.\n\n2. LPE specifier is part of the diagnosis of Conduct disorder. In the current paper it is mentioned that participants come from high-risk, difficult to access families. However it is useful to inform readers if those children exhibit symptoms that lead to the diagnosis of CD or any other comorbidity.\n\nMethod:\n3. In the participants section it is mentioned that children's ages ranged between 3 -19 years old. In the next paragraph the descriptive information of target children are not consisted (it is mentioned a range of 5-18 years old). It should be clarified if target children are the actual participants and if this is the case then why there are different descriptive statistics and range of age.\n\n4. It is mentioned that 6 families were deemed unsuitable or declined. Authors should provide the reasons that lead to this decision (not in details).\n\n5. In CAPE 1.1 description it is stated that a child with a diagnosis of ASD was included in the study. I am not convinced that a child with autism can participate in the current study. Despite their similarities in behavior and in empathy difficulties, ASD and CU traits have different underlying mechanisms. Thus, authors should not use the data of this participant or justify the decision of including them.\nResults:\n6. A descriptive table will help readers to have a more clear idea of all basic demographics information.\n\n7. In figure 1, authors provide very useful information regarding the differences between target and non-target children. However, it is not mentioned in the introduction and method section that authors will investigate this. Thus, it should be stated as an aim of the study and also in both measures and participants section.\n\n8. Figure 4 (correlations) is very difficult to follow\nDiscussion:\n9. Discussion is well written and the limitation section discuss all issues that are raised (both in method and interpretation). However authors again do not mention anything about the non-target children.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "5257",
"date": "27 Feb 2020",
"name": "Luna Centifanti",
"role": "Author Response",
"response": "Thank-you for the opportunity to revise and resubmit our paper to F1000Research. We are very glad to hear that there was interest in our manuscript and an appreciation for the contribution made with regards to providing support for the utility and validity of the CAPE. We would like to thank the three reviewers for their insightful and constructive comments on the first submission. Below we have responded to their comments and hope to have addressed the concerns adequately. Reviewer 2 Introduction Authors provide very detailed information regarding CAPE 1.1 and also the aim of the study. However, since authors investigated the difference between non-target with target children on several measures, something that is not mentioned in the introduction, they should revise the aim section by adding this information. We agree with this point and have removed this part of the assessment. This is outlined in response to [Reviewer 1’s] comment 2 (a). LPE specifier is part of the diagnosis of Conduct disorder. In the current paper it is mentioned that participants come from high-risk, difficult to access families. However, it is useful to inform readers if those children exhibit symptoms that lead to the diagnosis of CD or any other comorbidity. Local authorities identified families to take part in the government schemes. As researchers were not involved in this process, we cannot comment on whether the children of those families exhibited symptoms. However, we do acknowledge that children exhibiting symptoms of conduct problems often come from high-risk, difficult to access families. Children who show serious conduct problems tend to come from high-risk backgrounds involving disorganized, unmotivated or disadvantaged families. Method In the participants section it is mentioned that children's ages ranged between 3 -19 years old. In the next paragraph the descriptive information of target children are not consisted (it is mentioned a range of 5-18 years old). It should be clarified if target children are the actual participants and if this is the case then why there are different descriptive statistics and range of age. Thanks for noticing this. The first age range of 3-19 was descriptive of all children under the scheme but not all children were included in the study, therefore, we have removed this information to make it clearer. It is mentioned that 6 families were deemed unsuitable or declined. Authors should provide the reasons that lead to this decision (not in details). Thank-you for pointing out that this was not clear, we have added the below information for clarification. These were unsuitable because either the caseworkers were themselves afraid of violence or the families were already non-compliant with the caseworkers efforts at intervention. In CAPE 1.1 description it is stated that a child with a diagnosis of ASD was included in the study. I am not convinced that a child with autism can participate in the current study. Despite their similarities in behavior and in empathy difficulties, ASD and CU traits have different underlying mechanisms. Thus, authors should not use the data of this participant or justify the decision of including them. Thank-you for this valuable input, we have now added in the below information to justify our decision for including children with ASD. We decided to do assessments using the CAPE regardless of potential diagnoses of Autism Spectrum Disorder (ASD), because research shows that some children have a ‘double hit’ of ASD and callous-unemotional traits (Pasalich et al., 2014) and the modest phenotypic overlap observed in social-emotional deficits is largely explained by shared genetic variance (O’Nions et al., 2015). Results A descriptive table will help readers to have a more clear idea of all basic demographics information. We agree with this comment and have therefore added a figure (Figure 1) to make the demographic information clearer. In figure 1, authors provide very useful information regarding the differences between target and non-target children. However, it is not mentioned in the introduction and method section that authors will investigate this. Thus, it should be stated as an aim of the study and also in both measures and participants section. To ensure the focus remains on the validation of the CAPE we have chosen to remove the assessment of the non-target youth. This is outlined in response to [Reviewer 1’s] comment 2 (a). Figure 4 (correlations) is very difficult to follow In light of this comment and [Reviewer 1’s] comment 6 (b), we have now presented the information in a correlation table. Discussion Discussion is well written, and the limitation section discuss all issues that are raised (both in method and interpretation). However, authors again do not mention anything about the non-target children. Thank-you for such positive feedback on our discussion. We have now removed the assessment of non-target children, as we agree the purpose for inclusion was not clear. This is outlined in response to [Reviewer 1’s] comment 1 (a)."
}
]
},
{
"id": "56052",
"date": "15 Nov 2019",
"name": "Timothy R. Stickle",
"expertise": [
"Reviewer Expertise Callous Unemotional Traits",
"Developmental Psychopathology",
"Co-occurring mental health and substance use disorders",
"Statistics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reports on a study of CAPE assessment for the limited prosocial emotions specifier to DSM-5 conduct disorder in a high-risk sample. Results make a contribution in that they provide support for the construct validity of the CAPE. Evidence for clinical utility is equivocal. There are a few areas for clarification that would help to better understand aspects of the study and its results.\n\nMethod/Sample The manuscript notes different age ranges. If I understand correctly the target children ranged in age from 5-18 years and that all children ranged in age from 3-19 years. I think it would help the reader to explicitly denote these age ranges in terms of target and comparison children (or some other fashion). Please comment on whether we should we expect the LPE specifier and thus the CAPE to perform similarly across this wide developmental range?\n\nThe total sample of children appears to be N = 85. This sample size should be stated explicitly as the manuscript states it somewhat indirectly in terms of describing a breakdown of 50 non-target and 35 target children when describing group characteristics.\n\nGiven that there are 34 families and, if I understood correctly, 85 children, ratings of children in the same family by the same parent are nested and so non-independent. The nesting calls into question the validity of p-values derived from tests of statistical significance assuming independence of observation. Some way of addressing this concern should be included.\n\nAn additional statistical concern, is low power, as acknowledged by the authors as a caveat to interpreting results. A formal power analysis should be included. This information will help the reader to better evaluate both significant and non-significant findings in terms of effect size and sample size.\n\nConclusions/Discussion The hypothesized utility of the LPE specifier is to identify children at higher risk for severe and persistent conduct problems and antisocial behavior.\n\nThe authors should address the utility of LPE/CAPE, given that no higher risk youth were identified in this sample. Some specific factors to address: The lack of identifying such a subgroup may be entirely due to insufficient statistical power. As noted above, asserting that low power led to this result requires a power analysis. It may also be that elevated risk may be evident over time rather than cross-sectionally.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5258",
"date": "27 Feb 2020",
"name": "Luna Centifanti",
"role": "Author Response",
"response": "Thank-you for the opportunity to revise and resubmit our paper to F1000Research. We are very glad to hear that there was interest in our manuscript and an appreciation for the contribution made with regards to providing support for the utility and validity of the CAPE. We would like to thank the three reviewers for their insightful and constructive comments on the first submission. Below we have responded to their comments and hope to have addressed the concerns adequately.Reviewer 3 Method/Sample The manuscript notes different age ranges. If I understand correctly the target children ranged in age from 5-18 years and that all children ranged in age from 3-19 years. I think it would help the reader to explicitly denote these age ranges in terms of target and comparison children (or some other fashion). Please comment on whether we should we expect the LPE specifier and thus the CAPE to perform similarly across this wide developmental range? This point was also raised by the second reviewer and we recognise that the information given is confusing. The first age range of 3-19 was descriptive of all children under the scheme but not all children were included in the study, therefore, we have removed this information to make it clearer. We have also included the below information in the Method section to address concerns regarding the use of the CAPE across a wide developmental range. The manual specifies that raters use their developmental psychology training to ensure that symptoms are rated with regard to what is typical behaviour for a child based on their age. Both raters had sufficient training in developmental psychology and the assessment of callous-unemotional traits to assess children as young as 2-3 years of age (Waller et al., 2016). The total sample of children appears to be N = 85. This sample size should be stated explicitly as the manuscript states it somewhat indirectly in terms of describing a breakdown of 50 non-target and 35 target children when describing group characteristics. Thank-you for this suggestion. We have added the below to make this clearer. One of the target children was selected at random from the family that had two target children. Therefore, the total sample of children (N=84), consisted of 34 target children and 50 non-target children. Given that there are 34 families and, if I understood correctly, 85 children, ratings of children in the same family by the same parent are nested and so non-independent. The nesting calls into question the validity of p-values derived from tests of statistical significance assuming independence of observation. Some way of addressing this concern should be included. We have made the decision to remove the analyses for the part of the assessment that includes the non-target children - outlined in response to [Reviewer 1’s] comment 2 (a). Therefore this is no longer an issue. An additional statistical concern, is low power, as acknowledged by the authors as a caveat to interpreting results. A formal power analysis should be included. This information will help the reader to better evaluate both significant and non-significant findings in terms of effect size and sample size. To address this we have added information about the relevant confidence intervals to the results section. Most confidence intervals were large, indicating that our estimations could be improved by a larger sample – which is something we later acknowledge in the discussion. Conclusions/Discussion The hypothesized utility of the LPE specifier is to identify children at higher risk for severe and persistent conduct problems and antisocial behavior. The authors should address the utility of LPE/CAPE, given that no higher risk youth were identified in this sample. Some specific factors to address: The lack of identifying such a subgroup may be entirely due to insufficient statistical power. As noted above, asserting that low power led to this result requires a power analysis. It may also be that elevated risk may be evident over time rather than cross-sectionally. In response to Reviewer 1, we added some discussion of the clinical utility of the CAPE in the Discussion section. We also added more discussion around power analyses and whether prospective designs might be useful. In addition, two publications have since been published so we compare our findings about high-risk youth to their studies of clinic-referred and offending youth samples."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1027
|
https://f1000research.com/articles/9-154/v1
|
26 Feb 20
|
{
"type": "Research Article",
"title": "An evaluation of a personalised text message reminder compared to a standard text message on postal questionnaire response rates: an embedded randomised controlled trial",
"authors": [
"Ann Cochrane",
"Charlie Welch",
"Caroline Fairhurst",
"Sarah Cockayne",
"David J. Torgerson",
"OTIS Study Group",
"Charlie Welch",
"Caroline Fairhurst",
"Sarah Cockayne",
"David J. Torgerson"
],
"abstract": "Background: Research outcome data is commonly collected using postal questionnaires; however, poor response can introduce bias and reduce statistical power. Text messaging is simple, cost-effective, and can be customised to the individual. Personalised, reminder text messages may improve response rates. Methods: A two-arm, parallel group ‘Study within a Trial’ (SWAT) was embedded within the Occupational Therapist Intervention Study (OTIS), a randomised controlled trial of a home assessment for falls prevention in older people. OTIS participants who provided a mobile phone number were randomly allocated (1:1) to receive either a personalised text message (Title, Surname, plus York Trials Unit (YTU) text) or the standard YTU text alone, prior to receiving their four-month post-randomisation follow-up postal questionnaire. The primary outcome measure was the proportion of participants who returned the questionnaire. Secondary outcomes were: time to response, completeness of response, requirement of a reminder letter, and cost-effectiveness. Binary data were compared using logistic regression and time to response by Cox proportional hazards regression. Results: A total of 403 participants were randomised: 201 to the personalised text and 202 to the standard text. Of the 283 participants included in the final analysis, 278 (98.2%) returned their questionnaire; 136 (97.8%) for the personalised text versus 142 (98.6%) for the standard text (adjusted odds ratio 0.64, 95% CI 0.10 to 3.88, p=0.63). The median time to response was nine days in both groups. In total, 271 (97.5%) participants returned a complete questionnaire; 133 (97.8%) in the personalised text versus 138 (97.2%) for the standard text. In total, 21 reminder letters were sent. The additional cost of personalised text messages was £0.04 per participant retained. Conclusions: Personalised texts were not superior to standard texts in any outcome assessed in our study. Further SWATs are needed to perform a meta-analysis and obtain more evidence. Registration: ISRCTN22202133; SWAT 35.",
"keywords": [
"SWAT",
"Randomised Controlled Trial",
"personalised",
"SMS text",
"postal questionnaire",
"reminder"
],
"content": "Introduction\n\nEvaluating strategies to improve the efficiency of conducting trials is a priority. Achieving high response rates for postal follow-up questionnaires is challenging; non-response threatens study validity through bias and reduced effective sample size1. Rigorous evaluation can be achieved by undertaking a Study within a Trial (SWAT)2–4. A SWAT is a self-contained study embedded within a host trial, which aims to evaluate an intervention5.\n\nThere are many strategies towards improving response to postal questionnaires including short messaging service (SMS) text prompts; however, uncertainty remains6,7 as to their effectiveness8–13. Furthermore, some evidence exists14 to suggest that personalised texts, in which recipients were addressed by name, increased average payment of delinquent fines compared to non-personalised texts.\n\nHere, we report the results of a SWAT evaluating a personalised text compared to a standard (non-personalised) text on postal questionnaire response rates in an elderly population.\n\n\nMethods\n\nThis two-arm, parallel-group, individually randomised controlled trial (RCT) was embedded within OTIS, a UK-based modified cohort RCT of occupational therapist-led home environmental assessment for the prevention of falls in older people15. This SWAT was registered as part of the host trial (OTIS) registration (ISRCTN22202133; date registered: 20.06.2016) and with the Northern Ireland Hub for Trials Methodology Research SWAT Repository (SWAT 35; date registered: 20.02.2016).\n\nEligible OTIS participants who agreed to receive text communication during participation, provided a mobile number, and were due to receive their four-month post-randomisation postal questionnaire, were randomised into this SWAT.\n\nParticipants received a single text four days after their four-month questionnaire was posted (Table 1).\n\nThe primary outcome was the proportion of participants who returned their four-month postal questionnaire. Secondary outcomes were: time to response, completeness of response, use of a reminder letter, and cost-effectiveness (Table 2).\n\nAs is usual for embedded trials, no formal power calculation was undertaken3–5 as the sample size was constrained by the number of participants available in the host trial.\n\nEligible participants (n=403) were randomised (1:1) using randomly varying blocks of four and six, stratified by OTIS trial group allocation. Allocations were generated by the OTIS trial statistician using Stata version 13.0, before being shared with the YTU data management staff responsible for the setup of the text messaging system. Eligible participants were then matched against the generated sequence in the order that they were randomised to the main trial.\n\nParticipants were not aware of their involvement within this SWAT; only to the OTIS trial group allocation. Study team members performing administrative, statistical or health economic roles were also not blinded, but data entry staff were.\n\nApprovals were granted by NHS West of Scotland Research Ethics Committee 3 (ref. 16/WS/0154); the University of York, Department of Health Sciences Research Governance Committee and the Health Research Authority. Consent for the SWAT was waived by the above-named Research Ethics Committee.\n\nAnalyses were conducted in Stata version 15.016. Baseline characteristics are summarised descriptively (Table 3). Binary outcomes were analysed using logistic regression, and time to questionnaire return was analysed using Cox proportional hazards regression. Time to return was truncated at 120 days allowing for the next follow-up time point (eight months post-randomisation) and illustrated using a Kaplan-Meier curve. Models were adjusted for SWAT and OTIS trial allocation. Unadjusted analyses of both binary and time to event outcomes are also presented. The costs incurred retaining participants are summarised descriptively (Table 5).\n\n*0-worst health you can imagine, 100-best health you can imagine\n\n\nResults\n\nDelays setting-up the text messaging system meant no texts were sent prior to 7th December 2017. In total 120 (29.8%) randomised participants were due texts before this date. These participants are therefore excluded from the analysis. Participants (n=283) due texts on or after this date were analysed as randomised (Figure 1).\n\nResults are presented in Table 4. A total of 136 (97.8%) participants in the personalised text group returned their four-month questionnaire, compared with 142 (98.6%) in the standardised text group (adjusted odds ratio (OR) 0.64, 95% CI 0.10 to 3.88, p=0.63). In total, 10 personalised text participants were sent a reminder letter and 11 in the standard text arm. Of 278 returned questionnaires, 271 (97.5%) were completed: 97.8% in the personalised arm and 97.2% in the standard text arm (adjusted OR 1.29, 95% CI 0.28 to 5.89, p=0.75).\n\n* Primary\n\nThe median time to return was nine days in both groups. A log-rank test gave a p-value of 0.57; hence, the data provide little evidence to reject the hypothesis that the two groups have the same survival function. The Cox proportional hazards model corroborated this (hazard ratio 1.06, 95% CI 0.84 to 1.35, p=0.60) (Figure 2). Examination of the log-log plots of the estimated survival functions, and a global test of the Schoenfeld residuals suggested the proportional hazards assumption was reasonable (p=0.52).\n\nStandard texts were 159 characters (costing £0.048), whereas personalised texts ranged from 166 to 178 characters (costing £0.096). Other costs included reminder letters and additional questionnaires posted to non-responders (£2.35 each) (Table 5).\n\n\nDiscussion\n\nThese results provide little support to the hypothesis that personalisation of texts improves postal questionnaire return rate compared to standard texts, in this population. There was also little evidence to suggest that personalisation led to quicker returns of questionnaires, improved questionnaire completion, or reduced the requirement for a reminder letter to elicit a response. The additional cost of personalised texts was £0.04 per participant retained.\n\nEligible participants who provided a mobile phone number at enrolment to the host trial (78.4%) was lower than antipated. Nearly 30% of SWAT participants had to be excluded from analysis due to problems with text automation. Furthermore, the high proportion of returned postal questionnaires in the standard text group meant only very small improvements could ever be observed or that a ceiling effect may have been reached. Thus, a large sample size would be required in order to provide strong evidence against the null hypothesis in favour of personalisation. Together, the small sample size and high baseline event rate mean this SWAT provides limited evidence for (or against) the personalisation of texts as a means to improving retention of participants.\n\n\nConclusions\n\nGiven the uncertainty regarding the effectiveness of personalising text messages, we feel that further investigation via RCTs is warranted. Meta-analysis could be used to obtain a more precise estimate for the effectiveness of personalising texts and explore variation across different participant characteristics.\n\n\nData availability\n\nOpen Science Framework: OTIS Trial Text SWAT. https://doi.org/10.17605/OSF.IO/KH75X17.\n\nThis project contains the following underlying data:\n\nOTIS_textswat_data (CSV). Underlying data associated with this study.\n\nOTIS_textswat_data (DTA). Underlying data associated with this study.\n\nOTIS_textswat_data_key (CSV). Key to abbreviaitons used in dataset.\n\nOpen Science Framework: CONSORT checklist for ‘An evaluation of a personalised text message reminder compared to a standard text message on postal questionnaire response rates: an embedded randomised controlled trial’. https://doi.org/10.17605/OSF.IO/KH75X17.\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 also like to thank the embedded trial participants who returned trial questionnaires.\n\nThis paper was written by the authors on behalf of the OTIS Study Group, which is made up of the following individuals: Sophie Boyes (York Teaching Hospital NHS Foundation Trust); Sarah Cockayne (University of York); Shelley Crossland (Leicestershire Partnership NHS Trust); Avril Drummond (University of Nottingham); Caroline Fairhurst (University of York); Simon Gilbody (University of York); Catherine Hewitt (University of York); Sarah E Lamb (University of Exeter); Jennifer McCaffery (University of York); Alison Pighills (Mackay Base Hospital; Mackay Australia and James Cook University); Clare Relton (Barts and The London School of Medicine and Dentistry); Sara Rodgers (University of York); Sarah Ronaldson (University of York); Katie Whiteside (University of York), and David J. Torgerson (University of York). With the exception of Ann Cochrane (University of York), who supported data collection for the host OTIS trial, all members of the OTIS Study Group were involved in designing this SWAT.\n\nThe authors acknowledge Adwoa Parker (University of York) for pre-registering this embedded trial with the Northern Ireland Hub for Trials Methodology Research SWAT Repository (SWAT 35).\n\n\nReferences\n\nTorgerson D, Torgerson C: Designing Randomised Trials in Health, Education and the Social Sciences. Basingstoke: Palgrave MacMillan; 2008. Publisher Full Text\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials. 2014; 15(1): 399. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdamson J, Hewitt CE, Torgerson DJ: Producing better evidence on how to improve randomised controlled trials. BMJ. 2015; 351: h4923. PubMed Abstract | Publisher Full Text\n\nMadurasinghe VW; Sandra Eldridge on behalf of MRC START Group and Gordon Forbes on behalf of the START Expert Consensus Group: Guidelines for reporting embedded recruitment trial. Trials. 2016; 17: 27. 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(1): 139. 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; (3): 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; (12): MR000032. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshby R, Turner G, Cross B, et al.: A randomized trial of electronic reminders showed a reduction in the time to respond to postal questionnaires. J Clin Epidemiol. 2011; 64(2): 208–212. PubMed Abstract | Publisher Full Text\n\nKeding A, Brabyn S, MacPherson H, et al.: Text message reminders to improve questionnaire response rates. J Clin Epidemiol. 2016; 79: 90–95. PubMed Abstract | Publisher Full Text\n\nClark L, Ronaldson S, Dyson L, et al.: Electronic prompts significantly increase response rates to postal questionnaires: a randomized trial within a randomized trial and meta-analysis. J Clin Epidemiol. 2015; 68(12): 1446–1450. PubMed Abstract | Publisher Full Text\n\nMan MS, Tilbrook HE, Jayakody S, et al.: Electronic reminders did not improve postal questionnaire response rates or response times: a randomized controlled trial. J Clin Epidemiol. 2011; 64(9): 1001–1004. PubMed Abstract | Publisher Full Text\n\nBrabyn S, Adamson J, MacPherson H, et al.: Short message service text messaging was feasible as a tool for data collection in a trial of treatment for irritable bowel syndrome. J Clin Epidemiol. 2014; 67(9): 993–1000. PubMed Abstract | Publisher Full Text\n\nRichmond SJ, Keding A, Hover M, et al.: Feasibility, acceptability and validity of SMS text messaging for measuring change in depression during a randomised controlled trial. BMC Psychiatry. 2015; 15: 68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaynes LC, Green DP, Gallagher R, et al.: Collection of Delinquent Fines: An Adaptive Randomized Trial to Assess the Effectiveness of Alternative Text Messages. J Policy Analysis and Management. 2013; 32(4): 718–30. Publisher Full Text\n\nCockayne S, Pighills A, Adamson J, et al.: Can occupational therapist-led home environmental assessment prevent falls in older people? A modified cohort randomised controlled trial protocol. BMJ Open. 2018; 8(9): e022488. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStataCorp: Stata Statistical Software: Release 15. College Station, TX:StataCorp LLC 2017. Reference Source\n\nWelch C: OTIS Trial Text SWAT. 2020. http://www.doi.org/10.17605/OSF.IO/KH75X"
}
|
[
{
"id": "60604",
"date": "11 Mar 2020",
"name": "Frances Shiely",
"expertise": [
"Reviewer Expertise Epidemiology and Clinical Trial Methodology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 was a SWAT within the OTIS trial, an occupational therapist-led home environmental assessment for the prevention of falls in older people in the UK. The SWAT was a two-arm, parallel group study. The primary outcome was the proportion of participants who returned the four-month follow-up postal questionnaire. Secondary outcomes were: time to response, completeness of response, requirement of a reminder letter, cost effectiveness. The conclusion was that personalised texts were not superior to standard texts in any of the outcomes assessed. This is a very well conducted study and the clarity of presentation is to be commended.\n\nAnswers to main headings for the review:\nThe work is clearly and accurately presented for the most part, and it does cite the relevant literature as well as the current literature. I have added some comments/corrections below to address.\n\nThe study design is appropriate and the work is technically sound. Though I understand the purpose of the study was to evaluate personalised text messages versus non-personalised messages, I would have liked to see a comparison with those that received reminder letters only. Would this be possible with the current data set?\n\nSufficient details of the methods and analysis are provided for the most part but I’ve asked for a few minor issues to be addressed in the comments below.\n\nThe statistical analysis and its interpretation are appropriate.\n\nYes, the authors have added information on data availability.\n\nThe conclusions drawn are supported by the results but they are different in the abstract to the discussion section of the paper. The authors are definitive in the abstract but are less definitive in the discussion section saying the results provide “little” support. Can we really say little support? I think this should be stronger. There is no evidence to support personalised text messages in this study. I understand there were limitations but these are the findings from this study. I’d ask the authors to address the use of the term “little” in the discussion section.\n\nFurther Comments/Corrections:\nThe reminder letter appears for the first time in the results section and in table 4. This needs to be detailed in the methods section. What justified a person being sent a reminder letter/why were only 21 reminder letters sent? Also, you should add the reminder letter to Figure 1. I expect that their inclusion didn’t affect the results given the small numbers and equal proportions in both groups, however, a re-run of the analysis excluding them, and a sentence to say that it didn’t affect the results.\n\nRather than just present the actual costs of each type of text message in the results section under “cost-effectiveness”, can you present the findings of the analysis from both? I appreciate it’s presented in Table 5 but comment on it.\n\nThe outcome definition in Table 2 “proportion of questionnaires returned” should be adjusted as its current meaning does not match your intention. It is not the proportion of questionnaires returned to YTU at four months post-randomisation, it is the proportion of questionnaires returned or the proportion of questionnaires distributed at the four-month randomisation period that were returned or as you have in your main text, the proportion of participants who returned their four-month postal questionnaire. It would be helpful if you specified here how long you gave them to return it. I would add…\"within x days\".\n\nParagraph two of the results section, \"…compared with 142 in the standardised text group….” - replace standardised with standard.\n\nIn Table 5, the heading cost of texts is misleading as it implies that more than one text was sent to each person. Change to “cost of text”.",
"responses": []
},
{
"id": "61887",
"date": "21 Apr 2020",
"name": "Phil J. Edwards",
"expertise": [
"Reviewer Expertise Evidence-based Data Collection"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt has been shown previously that postal questionnaire response can be increased by using an SMS reminder (Edwards et al., 20091) and that questionnaire response can be increased by personally addressing participants by name on cover letters (Scott and Edwards, 20062). There have, however, been few randomised trials of a personalised reminder SMS. This study is therefore a welcome addition to the literature.\nThis is a report of the results of a randomised controlled trial nested within an RCT of occupational therapist-led home environmental assessment for the prevention of falls in older people. The nested trial sought to evaluate the effect on questionnaire response of a personalised text message compared to a standard (non-personalised) text in this elderly population. 403 participants who had agreed to receive text communication during participation, had provided a mobile number, and were due to receive their four-month post-randomisation follow-up postal questionnaire, were randomised into this nested trial. The proportion that responded in the personalised text message group was 97.8%, lower than the proportion that responded in the non-personalised text message group (98.6%); there was thus no evidence that the intervention had any effect on questionnaire response (p=0.63). The report is a clear and concise account of the study, citing the current literature. The authors may consider it helpful to include Pippa Scott’s study in their introduction or discussion. The study design seems appropriate and the work appears to be technically sound. The conclusions are adequately supported by the results, and the study makes a useful contribution to the data collection literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? 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/9-154
|
https://f1000research.com/articles/9-153/v1
|
26 Feb 20
|
{
"type": "Research Article",
"title": "Clinical and economic benefits of the Intellispace Portal applications CT TAVI and Spectral CT: Findings from a two-step Delphi panel",
"authors": [
"Mahdi Veillet-Chowdhury",
"James E. Harvey",
"Peter S. Wang",
"Saswat Panda",
"Mark Connolly",
"Marlene A. Mesarosh",
"Goran Medic",
"Jiwan Beth",
"Bodo Wiegand",
"Raman Danrad",
"Mahdi Veillet-Chowdhury",
"James E. Harvey",
"Peter S. Wang",
"Saswat Panda",
"Mark Connolly",
"Marlene A. Mesarosh",
"Goran Medic",
"Jiwan Beth",
"Raman Danrad"
],
"abstract": "Background: IntelliSpace Portal (ISP) is an advanced visualization platform that offers a single integrated solution designed to help physicians work more efficiently. This study aimed to develop a consensus statement on the clinical and economic benefits of two Intellispace Portal (ISP) applications (computed tomography (CT) transcatheter aortic-valve implantation (TAVI) and Spectral CT) applications. Methods: A modified Delphi method of two rounds of queries was used in a panel of 17 experts (11 for Spectral CT and 8 for CT TAVI). The experts were identified via referral and selected to achieve a balanced representation of subject matter experts. The Delphi process was conducted online using the MESYDEL platform. Two rounds of Delphi queries were conducted between July 22 and October 14, 2019. All data was collected prospectively. Results: Areas of consensus represented contribution of ISP applications toward quadruple aim goals. Consensus was reached that the Spectral CT application led to more confident decision making, improved diagnosis capabilities leading to better treatment paths, and a better work experience. Panelists noted that they required fewer tests to make a diagnosis using Spectral CT. The majority of physicians (63%) agreed that CT TAVI led to time savings for ‘device sizing’ results, and 75% of physicians agreed that the tool resulted in a better work experience. Conclusions: The increased diagnostic confidence of spectral information could eliminate the need for many follow-up scans in patients, resulting in clinical benefit to the patient in terms of reduced exposure to radiation and economic benefit to healthcare systems in reducing the number of additional scans. The staff satisfaction goal of the quadruple aim is also satisfied by Spectral CT and CT TAVI leading to a better work experience in our panelist physicians. This may result in indirect benefits such as reduced rates of physician burnout and greater provider satisfaction.",
"keywords": [
"Dual-energy CT",
"Spectral CT",
"TAVI",
"TAVR",
"IntelliSpace Portal"
],
"content": "Introduction\n\nIn recent years, healthcare spending in OECD countries has grown by an average of 1.8% in 2017—a noticeable decrease from the 3.5% growth observed in 2015 and 2016 and significantly below the rates experienced before the 2008 financial crisis (OECD, 2019a). Preliminary estimates for 2018 from the OECD point to growth having picked up to around 2.5% in 2018 (OECD, 2019a). At this rate, the rise of annual healthcare spending is above annual inflation rates (Barlas, 2019).\n\nOn average, health spending as a share of GDP was 8.8% in 2017. In the United States (U.S.) 17.1% of GDP is spent on healthcare, which is significantly more than Switzerland (12.3%) and France (11.3%), the second- and third-highest spenders (OECD, 2019a; OECD, 2019b). By comparison Turkey (4.2%), Luxembourg (5.4%) and Mexico (5.4%) were the lowest spenders in terms of percentage of GDP (OECD, 2019a; OECD, 2019b). Around 75% of health spending on average in OECD countries is paid through government or compulsory insurance arrangements (OECD, 2019a).\n\nBecause the current rising healthcare costs are unsustainable for governments in the long-run, Donald Berwick and colleagues at the Institute for Healthcare Improvement (IHI) provided a framework for the delivery of high value care in the USA—the Triple Aim. Triple Aim is centered around three overarching goals: improving the individual experience of care; improving the health of populations; and reducing the per capita cost of healthcare simultaneously (Berwick et al., 2008). Health systems globally grapple with the challenge of improving the health of populations while simultaneously maintaining or lowering healthcare costs (Sikka et al., 2015).\n\nThe successful achievement of the Triple Aim requires highly effective healthcare organizations. The backbone of any effective healthcare system is an engaged and productive workforce (LLI, 2013). However, the Triple Aim does not explicitly acknowledge the critical role of the workforce in healthcare transformation. This is why the ‘quadruple aim’ has become a new and important framework by adding a fourth component to the equation: improving the staff experience (Bodenheimer & Sinsky, 2014; Jeffs, 2018; Sikka et al., 2015).\n\nIntelliSpace Portal (ISP) (Philips, Amsterdam, The Netherlands) is an advanced visualization platform that offers a single integrated solution designed to help physicians work quickly, especially for complex cases and follow-up. Key features provided by ISP are the following: (1) to provide a comprehensive overview of the patient and to quantify and diagnose using multi-modality clinical applications accessed from any point of hospital’s network; (2) to provide a broad range of leading clinical applications that span multiple domains; (3) to enable a physician to focus more on patient care by creating workflow efficiencies and time-saving tools that have been shown to cut complex patient analysis time by up to 77% (Kadavigere et al., 2011) and automatically adapt to the physician’s way of working (Philips, 2016b; Philips, 2018).\n\nISP leverages a broad range of over 80 applications, spanning clinical domains including oncology, cardiovascular, neurology, pulmonology and others. These applications offer exceptional flexibility to access, analyze, and quantify patient data in one unified view (Philips, 2016b; Philips, 2018). Under the rubric of quadruple aim in healthcare, we explore the clinical and economic benefits of two selected ISP applications: computerized tomography (CT) transcatheter aortic valve implantation (TAVI) and spectral CT.\n\nCT TAVI is a non-invasive post-processing application that provides semi-automatic measurements of the aorta and aortic valve that are necessary for effective pre-TAVI planning. Specifically, the software provides model-based segmentation of the aortic valve, ascending aorta, and left ventricle with automatic calcium segmentation, improved landmark detection, and semi-automated detection of the coronary ostia. In addition, it provides semi-automated detection and dimensions measurements of the aortic annulus, left ventricular outflow tract, sinotubular junction, sinus of valsalva, ascending aorta and distance to coronary ostia for TAVI-device sizing. It also predicts an accurate starting angle of the C-arm for device deployment in the catheterization laboratory or hybrid operating room. Lastly, it can provide a semi-automated assessment of iliofemoral and subclavian vascular access, thus enabling additional potential time savings (Philips, 2016b; Philips, 2018).\n\nThe ISP suite of Spectral CT applications has been optimized for the viewing and analysis of spectral data sets from the IQon Spectral CT scanner (Philips, Amsterdam, The Netherlands). Physicians can access the desired applications on the virtual desktop via thin client from anywhere in the hospital and also from outside the hospital via a secured network connection. The tools help radiologists improve tissue differentiation, quantify iodine concentration, and support diagnoses. The clinical benefits of spectral applications include: (1) Enhancement of the conventional image by overlaying an iodine map; (2) Determining the presence or absence of enhancement via virtual non-contrast images; (3) Visualization of images at different energy levels (40–200 keV); (4) ease of switching between various spectral results through a viewport control; (5) the ability to manage and create user/site-specific presets; (6) the ability to characterize a lesion using scatter plots; and (7) Tissue characterization using attenuation curves. The biggest economic benefits are offered by reduction of follow-up exams (Philips, 2016a; Prabhakar et al., 2018).\n\nThe objective of this research is to use an online Delphi survey framework to gather information on real world use and develop a consensus statement on the clinical and economic attributes of two ISP applications: CT TAVI and Spectral CT.\n\n\nMethods\n\nThe Delphi method is a structured forecasting/decision-making tool that has been successfully applied to health research in many disciplines (Balabanova et al., 2011; Booth et al., 2011). The Delphi method creates conditions that are favorable to a convergence of opinions, while at the same time allowing moderators to clearly discern points of dissent. It usually takes the form of a written questionnaire and allows for anonymous and independent consultation and argumentation, thereby avoiding some of the drawbacks of face-to-face confrontations on both the social level (e.g. power relations within a group) and the practical level (time consuming, especially with geographically dispersed individuals, which is also applicable to our case) (Duin, 2016). Responses are only visible to the moderator(s) and not to the participants in order to avoid self-moderation bias. The iterative nature of the consultation builds on the feedback of the respondents. This allows for the correction of potential bias in the initial questions which is the main fragility of classical (non-iterative and non-interactive) queries. A potential problem with the Delphi method is the creation of a bottleneck towards convergence of opinions during the process (Linstone & Turoff, 2002; van der Gracht, 2012). It is therefore important to use both open and closed questions, and to take into consideration the whole spectrum of opinions. The online platform used in our study provides additional advantages such as low respondent drop-out rates (reducing effects of self-selection) and the possibility for respondents to revisit, complement or modify their answers during both rounds (generating more data) (Francois et al., 2011).\n\nExpert users of Philips ISP - CT TAVI and Spectral CT were identified via referral by Philips clinical workflow specialists and selected to achieve a balanced representation of these expert users from different regions within the US. Experts were recruited via email. All experts declared all conflicts of interest before participating in the panel. Throughout the Delphi, respondents were encouraged to explain their choice for closed-ended and multiple-choice questions. All questions are available as part of the Underlying data (Wiegand, 2020a).\n\nThe Delphi process was conducted online using the MESYDEL platform (Mesydel, 2019) developed by the Spiral Research Center at the University of Liège (Belgium); however, it could readily be performed manually. Two rounds of Delphi queries were launched and analyzed between July 22 and October 14, 2019. The project organizers designed questions on the survey. A duration of six weeks for Round 1 and five weeks for Round 2 were given to panelists to respond to the surveys. Reminder emails were circulated for each iteration.\n\nThe second round mainly focused on ambiguities or disagreement in the first round, clarifying areas of disagreement, and fostering more consensus on approaching the issue differently according to the participants’ input. This was achieved by providing the survey responses from Round 1 to the panelist during Round 2 in which clarification or consensus was sought.\n\nQuantitative data for closed questions was tabulated using MESYDEL, an online Delphi panel platform, and Microsoft Excel 365. The responses to the first round of questioning informed the design of the second round of questions, thus allowing for questions to be modified as needed. Particular attention was paid to areas of disagreement. Additionally, questions were reviewed with the survey group based on key observations from round 1 of the survey. All data was collected prospectively and analyzed using Microsoft Excel to obtain descriptive and summary statistics.\n\nThe Delphi panel consisted solely of U.S.-based experts. A response rate of 73% was achieved with 11 radiologists responding to the Spectral CT portion of the survey and 80% for the CT TAVI portion of the survey with 8 physicians responding. Of the Spectral CT survey participants, 91% had over 10 years of experience and 55% had over 20 years of experience (Table 1). Of the participants in the TAVI survey, 87% had over 10 years of experience, and 50% had over 20 years of experience (Table 2).\n\nISP, IntelliSpace Portal.\n\nISP, IntelliSpace Portal.\n\nThe results described here were derived using the Delphi Panel approach which is based on expert opinion. No patients have been used for this study and no patient data is collected and no patient samples or results have been used. No individual can be identified from the results presented. No animals have been used in this study. All participants provided written informed consent to participate.\n\n\nResults\n\nResponses to each question from each participant are available as Underlying data (Wiegand, 2020a).\n\nThe sentiment toward the Spectral CT tool within the ISP was very positive, with 90% of the respondents agreeing (hereafter, “agreeing” refers to “somewhat agree” or “completely agree” responses to the Likert-style questions) that it clinically benefits their everyday work (Figure 1). The respondents qualified their answers with comments and three respondents noted the virtual non-contrast (VNC) test feature as the most relevant feature. In addition, three respondents commented that Spectral CT improves their diagnostic confidence (Underlying data, Question 4; Wiegand, 2020a). In a question to assess whether the Spectral CT tool has led to more confident decision-making, 100% of respondents agreed with this statement. One respondent stated that the confidence in decision-making is, “…a result of additional precious information in [the] form of Spectral Detector CT reconstruction, [which is] useful in many situations like evaluating indeterminate cysts and incindentalomas that are routinely encountered.” Other respondents indicated that they valued the VNC feature for use in stroke patients and that they use the additional image types to confirm diagnoses (Underlying data, Question 12; Wiegand, 2020a).\n\nQuestions were asked in a Likert-form, e.g., statements were preceded by: “Please state to what extent you agree with the following statement:”. The comparator to Spectral CT is not having Spectral CT or ISP. For Spectral CT questions, n = 11, for CT TAVI questions, n = 8. CT: computed tomography; TAVI: transcatheter aortic-valve implantation.\n\nAll participants agreed that the Spectral CT application has led to improved diagnostic capabilities, leading to better treatment paths for patients (Underlying data, Question 10; Wiegand, 2020a). They state that it helps them with certain types of patients such as stroke patients and those with pulmonary emboli and that the additional imaging lends to more confidence in diagnosing. This confidence is highlighted in an open-ended question, “What elements of Spectral CT do you feel lead to a better patient experience?” (Underlying data, Question 36; Wiegand, 2020a), where doctors elaborated on how confidence in diagnosis impacts patients. One doctor wrote: “Patients may not directly experience this but when a radiologist hedges it significantly changes the arc of their care. On the other hand, when a radiologist is more confident, then the clinician is also more confident putting the patient on anticoagulation.”\n\nIn a question to gauge the effect of the Spectral CT application on the work experience (Underlying data, Question 9; Wiegand, 2020a), 91% of doctors agreed that the application improved their work experience. Two doctors further qualified their statements by reiterating that diagnostic confidence is improved. Another doctor elaborated further and stated that, “with [Spectral CT] and its reconstructions we are now telling the complete story of the disease rather than just providing the snapshot with conventional CT. For me this leads to a pleasant and better work experience.” In Round 2 in a follow up question to delve deeper into the technologies impact on the work experience, we explored further for those participants that agreed in Round 1. Responses ranged from specific tools provided in the Spectral CT application such as, “better visualization of enhancement by low keV images and iodine mapping”, and the option to always have a retrospective picture archiving and communication system (PACS), as well as the virtual desktop link to easily collaboratively review and analyze data. They also note the benefit of being able to bookmark and share certain data amongst the clinical team. Another doctor gave a similar comment, but also noted that they believe that the tool may prevent additional follow up exams by allowing them to make a confident diagnosis. Panelists also mention that an improved work experience may help them counter provider burnout (Underlying data, Question 26; Wiegand, 2020a).\n\nWe also asked the panelists questions on the potential of Spectral CT to reduce the number of scans needed for diagnosis, “In Round 1 of the Delphi Panel a participating expert highlighted the following, please state to what extent you agree with the following statement: Spectral CT has helped improve on accuracy and confidence of diagnosis (compared to not having Spectral CT & ISP), leading to less frequent follow-ups and repeat exams.” (Underlying data, Question 33; Wiegand, 2020a). In total, 27% of respondents somewhat agreed, and 56% completely agreed, while 18% of them completely disagreed with this statement. The experts who did agree feel it resulted in time-savings gave estimates ranging from 15 minutes per scan, 30–60 minutes per day, and 40–50% time reduction. While the rest of the respondents to the open question asking for comment replied that they did not have an accurate sense of how much time was saved.\n\nThe CT TAVI application of the ISP was thought to yield time-savings in ‘device sizing’ related tests. A question based on this was asked to the experts and 85% agreed that it did reduce time-savings, while 15% responded “neutral” (Underlying data, Question 66; Wiegand, 2020a). There was a lack of responses to the open-answer portion of this question, but one piece of critical feedback was that “while the software may reduce work, the time for analysis is roughly the same”. Another respondent answered that “CT TAVI produces multiple measurements quickly with a small amount of user input.” In a follow-up question (Underlying data, Question 67; Wiegand, 2020a) under this topic, we asked for numerical estimates of time-savings. Only five of the respondents responded to this question, but of the four responses which were given in time units “per case”, they estimated that approximately 13.1 minutes are saved per case. One respondent estimated that they saved “1 to 2 hours per week”.\n\nWe also asked whether CT TAVI decreases one’s cognitive load due to automation in location selection within the tool (Underlying data, Question 69; Wiegand, 2020a) whereby 100% of the respondents agreed that it did reduce cognitive load. However, in the comments one expert qualified that, “the software reduces some of the workload, [but] the user still needs to check for errors, which are uncommon with the annular sizing tool, but common with the vascular analysis tool.” A further two experts noted that when the quality of the CT scan is good, annulus points and automated landmarks are accurate.\n\nQuestions pertaining to quadruple aim goals were presented to all participants in Round 1 of the survey. Some participants also received follow-up questions where clarification was needed in Round 2. Responses to these questions are summarized in Figure 2 for the Spectral CT portion of the survey and in Figure 3 for the CT TAVI portion of the survey.\n\n**This question only had 9 responses; Questions were asked in a Likert-form, and “Please state to what extent you agree with the following statement: The Spectral CT application has led to (compared to not having Spectral CT and IntelliSpace Portal (ISP)):” preceded each statement.\n\nQuestions were asked in a Likert-form, and “Please state to what extent you agree with the following statement: The CT TAVI application has led to (compared to not having CT TAVI and IntelliSpace Portal (ISP)):” preceded each statement.\n\n\nDiscussion\n\nThis study aimed to identify potential benefits of an advanced visualization platform, ISP, and its two applications: Spectral CT and CT TAVI. Areas of consensus represent the contributions of ISP towards the pillars of the quadruple aim methodology: improving the health of populations, enhancing the experience of care for individuals, reducing the per capita cost of health care, and higher levels of staff satisfaction. Consensus was reached that the Spectral CT application led to more confident decision making and a better work experience. While a consensus was not achieved for many points of the CT TAVI portion of the survey, there was a positive trend in the responses suggesting that the technology is beneficial and that it improves the work experience and effectiveness of patient treatment. The benefits of reproducible and accurate automated aortic annulus sizing and related TAVI planning measurements is potentially invaluable as TAVI planning is a multidisciplinary approach which allows subspecialists and vendors to make decisions on validated data.\n\nWe achieved consensus to the question on Spectral CT leading to improved diagnosis capabilities leading to better treatment paths for patients (Underlying data, Question 36; Wiegand, 2020a). A radiologist commented that confidence on their part leads to confidence on the clinician’s part on choosing therapies for the patient. This is done without hedging and thus removes the need for additional tests to give a diagnosis. Another doctor also noted that the ability to retrospectively perform spectral analysis on every patient is crucial because it removes the need to prospectively determine if a study requires spectral or dual energy analysis. They note, “the ability to perform the spectral analysis without a significant cost of additional radiation to the patient is also highly beneficial.” A key benefit of Spectral CT is this ability to retrospectively use data to create virtual unenhanced images and enhanced material density maps to glean more from a single scan (Massat, 2018). Because Spectral CT is a dose-neutral exam compared to conventional CT, the non-contrast phase of some protocols is eliminated and the need for additional exams is reduced (Massat, 2018). Spectral CT simultaneously uses two different energy settings, which allows differentiation of materials based on material density. Radiologists can use this feature of Spectral CT to acquire virtual unenhanced images as well as iodinated contrast material-enhanced material density images from a single scan (Silva et al., 2011). These features of Spectral CT improve patient care by minimizing their exposure to radiation dose from scans while also giving radiologists access to additional images to inform a diagnosis.\n\nDiagnostic imaging costs and use have increased dramatically over time. It was reported that CT use increased from 52 per 1000 enrollees in 1996 to 149 per 1000 in 2010 (Smith-Bindman et al., 2012). Additionally, approximately 20% to 50% of tests fail to provide information that improves clinical care (Neeman et al., 2012). Amidst burgeoning healthcare spending, it is imperative to investigate new technologies and interventions for reducing excessive tests. A key finding of our survey was that panelists felt that the use of Spectral CT improved their confidence in making a diagnosis. Additionally, they felt that they required fewer tests to make a diagnosis. We found that most doctors did not have an accurate sense of exactly how much time is saved from the implementation of image portals and the use of additional scans from Spectral CT (Underlying data, Question 33; Wiegand, 2020a). Availability of spectral information could eliminate the need for follow-up scanning in patients with sub-optimal exams and incidental findings, increasing the diagnostic confidence of radiologists and resulting in a potential annual savings of $55,000 or higher depending on the practice and number of exams (Prabhakar et al., 2018).\n\nParts of our survey were designed to measure the perceived amount of time-savings from the use of the ISP tool and Spectral CT. However, we found that most of the respondents did not have an accurate sense for how much time is saved. Some radiologists estimated time-savings of 30–60 minutes a day while others estimated 15 minutes per scan was saved. Other respondents noted that elimination of follow up procedures does not shorten their workflow and that more time is needed to process spectral data. These results show that the benefits of Spectral CT would be highly dependent on the exact practice and type of caseload of a radiologist.\n\nOf the eight physicians responding to the TAVI survey, five stated that they believed the tool lead to time savings when used for ‘device sizing’ results. Four of them gave their response in “per case” savings, giving a mean time savings of 13 minutes per case. The last doctor estimated a time savings of one to two hours per week from using the tool. Reducing time needed to conduct tests is crucial in improving radiologist throughput. Throughput of patients is complex and relies on many factors including patient availability, acuity, triage, and other activity (Levenson et al., 2012). Time-savings when reading any type of test can prove to be beneficial in improving throughput and volume of a practice.\n\nThe fourth aim of the IHI pertaining to staff satisfaction or “joy in work” was added because studies have shown that parameters such as physician burnout lead to lower patient satisfaction and even poorer patient outcomes (Halbesleben & Rathert, 2008). In a systematic review, researchers identified 21 of 30 studies that measured burnout found a significant association between burnout and patient safety, whilst a further four studies also found a connection to patient safety (Hall et al., 2016). Thus, an important result of our survey was that 91% of respondents agreed that the spectral CT application makes their work experience better. Doctors who gave comment also unanimously agreed that the remote work capabilities of ISP are very convenient and benefits them in sharing data and in continuity of work (Underlying data, Question 2; Wiegand, 2020a). They also noted that the technology enabled them to assist colleagues from home while providing second opinions for emergent cases through the virtual portal. Even a marginal increase in satisfaction amongst radiologists is significant as it would play some role in preventing burnout and dissatisfaction with work, which could potentially result in improving patient satisfaction, outcomes, and safety.\n\n\nConclusions\n\nAn expert panel found the Spectral CT and CT TAVI applications of the Intellispace Portal to be positive regarding many of the healthcare quality improvement goals outlined in the Quadruple aim framework. The panelists agreed that more confident diagnosis can be made with fewer scans and that they enjoyed using Spectral CT as a part of their practice. Further study utilizing real world experience is needed to determine potential economic and physician satisfaction benefits of these new tools in radiology.\n\n\nData availability\n\nFigshare: Extended data - Delphi Questions v1.0. https://doi.org/10.6084/m9.figshare.11734353.v1 (Wiegand, 2020a).\n\nThis project contains all questionnaires with all answers given by Delphi panel participants.\n\nFigshare: Extended Data - Delphi Panel ISP. https://doi.org/10.6084/m9.figshare.11778486.v1 (Wiegand, 2020b).\n\nThis project contains the multiple choice questions data used to generate Figure 1–Figure 3.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank the following members of the Philips ICAP team whose efforts facilitated the success of this document.\n\n1) Greg Woodward, Sr. Mgr. IntelliSpace Portal Implementation and Clinical Adoption\n\n2) Diane Gressley, Clinical Workflow Adoption Consultant, Project Lead\n\n3) Clinical Workflow Adoption Consultants; Evelyn Roehr, Diana Wisler, Robert Verity, Ryan Gunter\n\nDr. Raman Danrad:\n\nI. Speaker Bureau of Philips\n\nII. The contribution to article is personal opinion of the author and does not represent endorsement by LSUHSC and UMC- New Orleans, Louisiana USA.\n\n\nReferences\n\nBalabanova Y, Gilsdorf A, Buda S, et al.: Communicable diseases prioritized for surveillance and epidemiological research: results of a standardized prioritization procedure in Germany, 2011. PLoS One. 2011; 6(10): e25691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarlas S: Are Hospital Prices a Bigger Problem than Drug Prices?: Congress Doesn't Know, Doesn't Care. P T. 2019; 44(5): 259–299. PubMed Abstract | Free Full Text\n\nBerwick DM, Nolan TW, Whittington J: The triple aim: care, health, and cost. Health Aff (Millwood). 2008; 27(3): 759–769. PubMed Abstract | Publisher Full Text\n\nBodenheimer T, Sinsky C: From triple to quadruple aim: care of the patient requires care of the provider. Ann Fam Med. 2014; 12(6): 573–576. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBooth A, Clarke M, Ghersi D, et al.: Establishing a minimum dataset for prospective registration of systematic reviews: an international consultation. PLoS One. 2011; 6(11): e27319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuin P: Foresight in organizations: methods and tools. New York, NY: Routledge; 2016. Publisher Full Text\n\nFrancois A, Rieppi S, Thiry A: Démarches participatives et approaches quali-quantiatives: le logiciel Mesydel. La participation en action. Bern: Peter Lang. 2011. Reference Source\n\nHalbesleben JR, Rathert C: Linking physician burnout and patient outcomes: exploring the dyadic relationship between physicians and patients. Health Care Manage Rev. 2008; 33(1): 29–39. PubMed Abstract | Publisher Full Text\n\nHall LH, Johnson J, Watt I, et al.: Healthcare Staff Wellbeing, Burnout, and Patient Safety: A Systematic Review. PLoS One. 2016; 11(7): e0159015. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJeffs L: Achieving the Quadruple Aim in Healthcare: The Essential Role of Authentic, Complex and Resilient Nurse Leaders. Nurs Leadersh (Tor Ont). 2018; 31(2): 8–19. PubMed Abstract | Publisher Full Text\n\nKadavigere R, Maiya M, Rao V, et al.: Standardized Results of CT Angiography Obtained with Automated Postprocessing Using a Dedicated Server: A Workflow Optimization Study. A collaboration of Philips Healthcare and Kasturba Medical College at Manipal University, India. RSNA 2011 Scientific Assembly and Annual Meeting, November 26 - December 2, 2011, Chicago IL. 2011. Reference Source\n\nLevenson RB, Camacho MA, Horn E, et al.: Eliminating routine oral contrast use for CT in the emergency department: impact on patient throughput and diagnosis. Emerg Radiol. 2012; 19(6): 513–517. PubMed Abstract | Publisher Full Text\n\nLinstone H, Turoff M: The Delphi method: techniques and applications. Boston, MA: Addison-Wesley. 2002. Reference Source\n\nLLI: Lucian Leape Institute: Through the eyes of the workforce: creating joy, meaning and safer health care. Boston, MA: National Patient Safety Foundation. 2013. Reference Source\n\nMassat MB: Spectral energy is redefining CT imaging. Appl Radiol. 2018; 47(7): 28–33. Reference Source\n\nMesydel: Mesydel - Better Decisions Through Collective Intelligence. 2019; Accessed 15 July 2019. Reference Source\n\nNeeman N, Quinn K, Soni K, et al.: Reducing radiology use on an inpatient medical service: choosing wisely. Arch Intern Med. 2012; 172(20): 1606–1608. PubMed Abstract | Publisher Full Text\n\nOECD: Health expenditure. 2019a; [Accessed 19th July 2019]. Reference Source\n\nOECD: OECD Health Statistics 2019.2019b; [Accessed 19th July 2019]. Reference Source\n\nPhilips: Certainty lives in layers - Philips IQon Spectral CT. 2016a. Reference Source\n\nPhilips: IntelliSpace Portal 9.0 - Clinical Datasheet.2016b.\n\nPhilips: IntelliSpace Portal 10 - Clinical datasheet.2018.\n\nPrabhakar R, Rong R, Martinez-Rios C, et al.: Economic benefits offered by reduction of follow-up exams using spectral detector CT (SDCT). 2018.\n\nSikka R, Morath JM, Leape L: The Quadruple Aim: care, health, cost and meaning in work. BMJ Qual Saf. 2015; 24(10): 608–610. PubMed Abstract | Publisher Full Text\n\nSilva AC, Morse BG, Hara AK, et al.: Dual-energy (spectral) CT: applications in abdominal imaging. RadioGraphics. 2011; 31(4): 1031–1046. PubMed Abstract | Publisher Full Text\n\nSmith-Bindman R, Miglioretti DL, Johnson E, et al.: Use of diagnostic imaging studies and associated radiation exposure for patients enrolled in large integrated health care systems, 1996–2010. JAMA. 2012; 307(22): 2400–2409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Gracht H: Consensus measurement in Delphi studies Review and implications for future quality assurance. Tech Forcasting Soc Chang. 2012; 79(8>): 1525–1536. Publisher Full Text\n\nWiegand B: Extended data - Delphi Questions v1.0. figshare. Dataset. 2020a. http://www.doi.org/10.6084/m9.figshare.11734353.v1\n\nWiegand B: Extended Data - Delphi Panel ISP. figshare. Dataset. 2020b. http://www.doi.org/10.6084/m9.figshare.11778486.v1"
}
|
[
{
"id": "93130",
"date": "16 Sep 2021",
"name": "Alan Haycox",
"expertise": [
"Reviewer Expertise I am a recently retired Reader in health economics at the University of Liverpool Management School."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very much enjoyed reading the paper and feels that it adds significantly to the literature in this area. However to be of value as a scientific approach Delphi exercises need to be subject to the same rigour as any other area of research. It is general good practice to be transparent and to describe the methodology applied fully so that the results can be assessed in light of these factors. In this regard I would have welcomed additional information on two elements of the study firstly the selection ox experts who participated in the study and secondly the definition of 'consensus' and what steps if any were taken to explore dissenting views of participants. How representative were participants of the general population of clinicians who use ISP applications and exactly how did they ensure that an appropriate diversity of views were exhibited by the chosen experts? It would be helpful if the selection criteria could be outlined in the paper. Obviously the participants must have experience and expertise in the use of these techniques (the study wants to identify a correct answer rather than a unanimously agreed incorrect answer!) but within these constraints many clinicians would have met these criteria so how were participants actually chosen?\nIdentifying a definition of consensus in advance of a study's execution also enables the research to better identify dissent with outliers encouraged to put forward reasons for their opinion just in case the outlier actually proves to be correct or whether disagreements have been caused by a different interpretation of the questions and issues being addressed.\nHowever in general my concerns about the application of the Delphi technique are limited by the context and purposes underlying the study, the reasons for carrying it out and the purpose to which it is being put. The aim was to assess the potential benefits of ISP and its two applications in the context of the quadruple aim framework and in this aim I believe that it has generated a great deal of valuable data. I do however agree with the conclusion to the study that further study using real world data is warranted and needed to more fully address the benefits arising from these new radiological tools.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/9-153
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https://f1000research.com/articles/9-152/v1
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26 Feb 20
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{
"type": "Research Article",
"title": "How scientific research changes the Vietnamese higher education landscape: Evidence from social sciences and humanities between 2008 and 2019",
"authors": [
"Thi-Huyen-Trang Nguyen",
"Trung Tran",
"The-Tung Dau",
"Thi-Song-Ha Nguyen",
"Thanh-Hung Nguyen",
"Manh-Toan Ho",
"Thi-Huyen-Trang Nguyen",
"The-Tung Dau",
"Thi-Song-Ha Nguyen",
"Thanh-Hung Nguyen"
],
"abstract": "Background: In the context of globalization, Vietnamese universities, whose primary function is teaching, there is a need to improve research performance. Methods: Based on SSHPA data, an exclusive database of Vietnamese social sciences and humanities researchers’ productivity, between 2008 and 2019 period, this study analyzes the research output of Vietnamese universities in the field of social sciences and humanities. Results: Vietnamese universities have been steadily producing a high volume of publications in the 2008-2019 period, with a peak of 598 articles in 2019. Moreover, many private universities and institutions are also joining the publication race, pushing competitiveness in the country. Conclusions: Solutions to improve both quantity and quality of Vietnamese universities’ research practice in the context of the industrial revolution 4.0 could be applying international criteria in Vietnamese higher education, developing scientific and critical thinking for general and STEM education, and promoting science communication.",
"keywords": [
"Social sciences research",
"research capacity",
"scientific publication",
"STEM",
"open science",
"industrial revolution 4.0"
],
"content": "Introduction\n\nIn order to provide high-quality output, universities need to maintain their available inputs, including physical resources such as buildings, laboratories, and non-physical resources such as the research capacity of researchers. The performance of an organization can be assessed based on how it maintains and combines these resources (Wright & Snell, 1998). Although there have been many studies on research productivity at the national and institutional level, the question of individual faculty's research capacity internationally still needs to be investigated. In addition, the relationship between the research capacity of university lecturers and students’ achievement has been questioned. Currently, the task of improving research capacity for lecturers is still not an attractive investment option for universities, especially in developing countries where universities often put more importance on the teaching function of faculty members (Pham & Hayden, 2019; Salmi & Pham, 2019; Tran & Nghia, 2020). Around the world, organizations, and scientific circles in Asia, Africa, and even Europe still face obstacles in their scientific publication process (Flowerdew, 1999). The difficulties for researchers in Asia and Africa could be boiled down to technical issues such as language barriers, budget, and access to scientific journals; but also a lack of research skills in certain cases (Pho & Tran, 2016), while researchers in Western countries face problems in the thorough understanding and preparation of scientific publishing, ineffective criticism and the need for additional scientific research (Lyytinen et al., 2007). Lyytinen et al. (2007) have listed the most prominent barriers to research institutions in Europe as the following: language and writing skills; preparation of PhD-level human resources; organizational orientation on research; and financial resources. The competence of an organization depends largely on the sum of its individual members’ capacity for research; therefore, even the most prestigious organizations must devote themselves to the development and maintenance of personal research productivity, or at least remove barriers for their researchers.\n\nIn STEM fields, most barriers concern deficiencies in the infrastructure, e.g., laboratories and facilities (Manjunath & Shashidahra, 2011; Peppas & Harland, 1989), while social science researchers are more concerned with access to data, and research materials (Alzahrani, 2011; Mdemu et al., 2017). Considering the whole process of scientific research, university lecturers face many problems regardless of their disciplines. In particular, the lack of access to scientific resources is the most common obstacle (Canagarajah, 2002; Flowerdew, 1999), and has been described as the root of the difficulty of research problems (Duszak & Lewkowicz, 2008). During the research period, the most common difficulty is budget, language skills, and research skills (Brooks et al., 2005). Language skills, especially English proficiency, can also be considered as one of the common challenges for non-English-speaking researchers (Flowerdew, 1999). However, this obstacle is not only in the implementation stage but also extends to the publishing stage, when the researcher must work with the editorial board and reviewers to defend their arguments. The issue of ‘linguistic inequality’ must be addressed due to the high rate of rejection when submitting published articles and the dominance of English in science publishing (Clavero, 2010).\n\nIn Vietnam, there have been many programs to promote research and international publication in recent years, which are encouraged and supported at various levels, from nationwide government-initiated policies to directives within schools and institutes. Scientific publication count has become a significant criterion to assess the research capacity of university lecturers. However, most research is commonly conducted in separate specialized research institutes in Vietnam (Pham & Hayden, 2019). In Asia, according to the QS Asia University Rankings 2019, there are only two higher education institutions (in top 250): Vietnam National University in Hanoi (ranking, 124), and Vietnam National University in Ho Chi Minh City (ranking, 144). From rankings 250–500, there are five more universities: Hanoi University of Science and Technology (ranking, 261–270), Ton Duc Thang University (ranking, 291–300), Can Tho University (ranking, 351–400) and Hue University, University of Da Nang (ranking, 451–500). For research papers per faculty indicator, only Vietnam National University in Hanoi and Vietnam National University in Ho Chi Minh City are included in the ranking, respectively, 5.1 and 2.3 research papers per faculty.\n\nRegarding the international research network indicator, excluding Hue University and the University of Da Nang, the remaining universities are ranked as follows: Vietnam National University in Hanoi (104), reaching 74.2, with the lowest being Can Tho University (270), which reached 34. In particular, Ton Duc Thang University is the only university in Vietnam rated by the 4-star/5-star QS Stars University Rating, demonstrating the high level of international appeal, the quality of teaching, the level of research, and the learning environment. According to QS World University Rankings 2019, Vietnamese universities were rather modest in terms of rankings: the two national universities in Ho Chi Minh City were in the 701-750 group, whereas those in Hanoi were in the 801-1000 group.\n\nAccording to the THE World University Rankings in 2020, for the first time, Vietnam has three universities that made this prestigious world ranking, including Hanoi University of Science and Technology (801–1000), Vietnam National University in Hanoi (801–1000) and Vietnam National University in Ho Chi Minh City (1001+). Among these, it can be seen that the highest research rate (volume, income, and reputation) belongs to Vietnam National University in Hanoi (9.1), and the highest citations rate (research influence) belongs to Hanoi University of Science and Technology (42.3). These recent statistics suggest a great effort from Vietnamese universities to expand their influence internationally. The effectiveness of scientific investment is a central theme in the implementation of science-education projects for all policy, governance, and funding organizations (Vuong, 2018). However, in terms of research index, Vietnamese universities lagged behind other universities in the world, which is entirely not surprising as the criteria used in rankings – such as facilities, staff capacity, and research quality – remained weaknesses in Vietnamese universities (Hayden & Thiep, 2010). While Vietnam's research output has grown by about 17-20% a year (Manh, 2015; Nguyen, et al., 2017) in recent years, the country is not yet on par with leading research countries in the ASEAN region, such as Singapore, Thailand or Malaysia (Tuan & Ly, 2011). Moreover, the above figures cover all scientific fields in Vietnam, which are dominated by the natural sciences, which has published internationally earlier than social sciences and humanities (SSH).\n\nThis study aims to take a look at the SSH in Vietnam, a young field of research in terms of international scientific publications. By analyzing the overall trend in scientific practice in Vietnamese universities, the study discusses solutions to improve the efficiency of publications in SSH of Vietnamese universities in the context of the industrial revolution 4.0.\n\n\nMethods\n\nThe SSHPA (Social Sciences and Humanities Peer Award) database (http://sshpa.com/) was used to review the Vietnamese SSH research between 2008 and 2019. We chose 2008 because this was when the Vietnamese national funding agency National Foundation for Science and Technology Development (NAFOSTED) was established, equivalent to the United States’ National Science Foundation. The SSHPA database records the number of international publications of Vietnamese SSH researchers to review their scientific productivity. Here, international publications are defined as those published in ISI/Scopus-indexed journals, and journals that are recognized by the Vietnam national funding agency NAFOSTED (Decision no. 251/QĐ-HĐQL-NAFOSTED).\n\nMoreover, some of the descriptive analyses were also based on the 2018 Journal Impact Factor (JIF; Clarivate Analytics, Journal Citation Reports 2019), such as publication output.\n\nThe structure and technical specifications of the SSHPA database are publicly available in Vuong et al. (2018a). Some of the main features are tracking of individual scientists’ productivity, as well as their affiliations, and providing quick and descriptive statistics of total productivity.\n\nA data analysis website (http://sda.aisdl.com/) was used to generate descriptive figures and tables directly from the database. This is a closed access website; however, readers can recreate the figures and tables using Microsoft Excel or R statistical software based on the data that are publicly available (see Underlying data).\n\n\nResults\n\nAs of January 10, 2020, only 3138 scientific publications by 2002 Vietnamese authors were recorded in the SSHPA database. This means that the total scientific productivity of Vietnamese authors in SSH over the last 11 years is rather low compared to overall domestic research, which is around 26000 articles (Adams, et al., 2019).\n\nThe total annual output of SSH research by publications between 2008 and 2019 is shown in Figure 1. Initially, the growth rate is quite steady; then, from 2016, there is a marked boost in annual published output; from 2014 to 2018, the total output was nearly double compared to the previous six years. In 2019, the number jumped to 598, surpassing 2018 by 136 articles.\n\nData as of January 10, 2020, according to sshpa.com.\n\nBased on the 2018 JIF, a large proportion of Vietnam's international publications appear in journals with JIF < 2.5 (Figure 2). However, journals with JIF 3-5 also show a high number of publications from Vietnam. When the JIF is >5, the number of Vietnamese publications significantly decreases.\n\nData from Clarivate Analytics, Journal Citation Reports.\n\nTable 1 below lists the institutions in Vietnam that have published in journals with JIF ≥5 in 2018. In general, most national universities and institutions with strong research capacity have been able to produce publications in high JIF journals. However, high JIF publications are rare in Vietnam, with only 13 out of the total 462 articles in 2018 that are in journals with a JIF of above 5. Notably, Vietnam National University Hanoi and Vietnam National University Ho Chi Minh, which are among the most prestigious universities in the country, have publications in high impact journals. Similarly, the Vietnam Academy of Social Sciences also has articles in high impact journals.\n\nIn this era of globalization, Vietnamese universities are at the forefront of educational reforms through their research and scientific contribution. Currently, several universities, such as Nguyen Tat Thanh University, Duy Tan University, Ton Duc Thang University, and Phenikaa University, have an accelerating research output (See Table 1; Lich, 2019).\n\nEven though productivity is showing signs of progress in terms of publication count, the ratio of Vietnamese authors to published articles remains low (all under 1) (see Figure 3). The year 2019 is currently the highest, with almost 0.8 Vietnamese authors per article.\n\nDuring the entire ten years, from 2008 to 2018, the number of female authors in total participating in research and publication in the field remains smaller than the number of male authors (Figure 4).\n\nData as of December 14, 2019, according to sshpa.com.\n\nHowever, different developments could be observed when considering only new authors – defined as those who had their article published for the first time in the concerned year. In this case, the number of new female authors has increased steadily from 2008 to 2019, surpassing that of male authors from 2018 (Figure 5).\n\nData as of December 14, 2019, according to sshpa.com.\n\n\nDiscussion\n\nThe process of international cooperation in scientific research via co-authoring has seen a significant change over the last ten years (2008 to 2018) in Vietnam. In the context of integration and development, collaboration with fellow researchers abroad offers Vietnamese researchers an opportunity to experience international standards. Furthermore, given that universities and institutes in Vietnam are reorienting themselves towards research activities, it is inevitable that international publishing standards would soon take on an important role in evaluating academic results and scientific productivity. As such, international criteria for granting tenure tracks or appointing individuals into managerial positions should be implemented in Vietnamese universities. National policies have started to consider international standards when outlining the recruitment and appointment of university positions. Recent regulations on standards of scientific publication for doctoral candidates, doctoral instructors and professorship candidates may be referenced. Using international publishing as a criterion and guideline for standards of quality, domestic universities should also not neglect to develop internal strength, to ensure and promote productivity and efficiency requirements of scientific investment (Vuong, 2018).\n\nAnother change in the Vietnamese academic landscape, more specifically in the field of SSH, relates to gender equality. Recently, the subject of gender equality has gained much attention in Vietnam, especially as women continued to confirm their role in the workforce with the development of information and communication. Despite this, Vietnamese society still has certain conceptions about gender related to the division of labor, leading to career prejudices against women: namely, preventing women from entering jobs considered “strenuous,” and encouraging them to take up more people-centered jobs, such as those in healthcare or education (UNESCO & KWDI, 2013). Due to this stereotype and the crowd effect, young women often face more hesitation in choosing to follow the research path. The fact that men outweighed women in sheer accumulated numbers – in researchers as well as in publication point – is a manifestation of this phenomenon. A shortage of female scientists is a shortage of scientists in general; hence, creating a supportive environment to encourage women lecturers and scientists to have more opportunities in research would have the effect of improving the general scientific output in the country. As it has already been shown, the number of new female scientists had started to surpass that of new male scientists since 2018, suggesting Vietnamese women were ready to integrate into the academic environment and the publishing worlds. Overall, policies on wage transparency and on ensuring fairness in the voting and reviewing process for appointing titles and managerial roles will play an important role in achieving gender equality. It is also highly important to encourage female authors to participate in the editing process, comment on textbooks and curriculum, as well as raising teacher awareness about STEM and gender sensitization. The presentation of gender roles in the content of textbooks and learning materials must be done carefully, avoiding the over-focus on women's preferences and perspectives as it will increase gender prejudice that already exists (Fousyia & Musthafa, 2016). At the same time, while showing off the image of female students studying at STEM majors is essential to inspire students, designing these contents should also be prudent to avoid impression confusing and overwhelming STEM subjects with students (Rosenzweig & Wigfield, 2016). The UNESCO report also points out the impact of testing on students' STEM performance by gender. While boys are more likely to do well on standardized and multiple-choice tests (which may be due to a higher risk of inclusion), girls are more likely to favor essay and project writing because of language inclination and collaborative connectivity (Eurydice, 2010). In particular, the attitude of teachers might be gender-based and could affect female students, making the test results of this group lower while the male students hardly suffer the same effects (Beilock, et al., 2010).\n\nThe analysis of data on the academic community itself could be applied to improve the performance of scientific research and education in Vietnamese universities. Such analyses could consider taking advantage of Open Science trends and Open Data in particular. Open Science is viewed as one of the most critical trends in the current international academic scene, which creates profound effects on a global scale not only within academia but also socially and economically (Willinsky, 2005). The movement strives towards taking down paywalls that lock out scientific knowledge and resources, including scientific publications, scientific data, and research tools, from public domains. It suggests a radical transformation of the current \"closed\" state of science, which had prevailed for decades, even centuries; only those who paid could access the aforementioned academic materials, which entailed the restriction of knowledge to a relatively privileged group of people. With openness in science, not only would doing science and disseminating knowledge become more egalitarian between both individuals and polities, but academia would also become more transparent: society at large – and not only bureaucrats – would be able to hold scholars accountable for their use of the state budget, or even to cooperate with scientists. Open Science should be seen as a new approach in carrying out, conducting, and managing research rather than just a program with a definite term. It is also an opportune approach in the context of the so-called Industry 4.0, when data and digitalized information are increasingly considered as the most important resource. Industry 4.0 has, in fact, taken place globally and applied to the field of educational science in Vietnam and has since contributed to the improvement of efficiency and effectiveness of research in this field. In a well-known study named by Chan et al. (2005), the authors point out six benefits of Open Science and Open Data, including (i) expanded access to international research results; (ii) expanded access to research done in developing countries; (iii) improvement to the research results of universities; (iv) improvement in citations and impacts; (v) expanded access to scientific data; (vi) supported peer review. The availability of Open Data also helps increase transparency in research by making all data public. It also helps to accumulate knowledge in the sense that fellow scientists could easily follow up on future research suggestions in existing studies by reusing data to develop new research (Pampel & Dallmeier-Tiessen, 2014).\n\nSecondly, the academic community would benefit from rendering scientific research more accessible to mass audiences via science communication (SciComm). Efforts have been made to actively promote SciComm worldwide, attracting a large number of researchers or people who have gone through periods of professional or scientific research. It has, in fact, come increasingly close to becoming a professional job, which rapidly improved the quality of mass media dissemination of scientific knowledge. SciComm can also contribute to guiding the younger generation into a labor market more imbued by computer technology and data science than ever before.\n\nIn fact, letting students approach the scientific way of thinking and scientific methods from the high school level can create a premise to gradually improve the research capacity of the entire country. In recent years, STEM has emerged as an educational solution to the challenges of human resource quality in the 4.0 era, in which a large number of industries were replaced by automation. To improve STEM-related research and education capacity with interdisciplinary and cross-disciplinary integration, the general education program should consider focusing on nurturing systematic thinking, critical thinking, and creative skills.\n\nDatabases, analytical methods, and science communication have become essential factors that need to be implemented on many platforms. As the scope of what does constitute as data grows larger and data gathering methods become more and more efficient, data has become an important asset, both to private and national entities. Artificial intelligence, for example, is one creation that both stemmed from and benefitted from the enormous amount of data that had become storable and available. Databases, especially pertaining to science, health, and education could be considered precious resources at many levels and in various domains. Such data would give governments the necessary input to ensure efficient policy making and would allow researchers to further study social tendencies and issues. On a smaller scale, one such project of data gathering and storage was the Network of Vietnamese Scientists in Social Sciences and Humanities Project (https://sshpa.com). The result is rather significant: data extracted from this project had been utilized in seven publications (see Ho, et al., 2017a; Ho, et al., 2017b; Ho, et al., 2017c; Vuong, et al., 2017; Vuong, et al., 2018c; Vuong, et al., 2019c; Vuong, et al., 2018a).\n\nFinally, data storage is also an innovative and sustainable way to preserve cultural relics – such as ancient stories and old architecture – against the erosion of time. Modern approaches to statistical analysis such as the Bayesian method, can provide a stable fulcrum for scientists to harness and exploit such data (La & Vuong, 2019). In fact, a number of studies using Bayesian techniques have uncovered unique cultural phenomena based on folklores (Vuong et al., 2018) or old Hanoi houses (Vuong et al., 2019). Currently, as the frequentist approach to statistics is facing rigorous evaluation in the world, the use and mastery of Bayesian statistics would certainly be precious skills to gain for a nascent academic community such as that of Vietnamese SSH scholars.\n\n\nConclusion\n\nEncouraging scientific research and improving productivity in terms of publication count, more specifically international publications, are urgent requirements to improve the performance of Vietnamese universities, especially in SSH. Additionally, promoting international integration through cross-border scientific collaboration could greatly improve the nation’s technology, as well as fostering a solid foundation for the development of Vietnam's education system. In terms of economic development, while the facilities of Vietnamese universities have been improved in the past years, the quality of human resources and research capacity remained in the development stage. Overall, scientific investment in Vietnam is often seen as wasteful in the public eyes because there is a lack of quality control, and lengthy transition from theory to practical application. The situation of scientific publication in Vietnam is particularly modest when it comes to SSH: not only is the number of SSH faculties actively doing research still limited, contributions are also concentrated on a small number of researchers. In the context of globalization and development, enhancing international cooperation is a specific direction that Vietnamese researchers would greatly benefit from, in order to adapt to international standards. However, domestic universities should not neglect internal improvement to ensure and promote quality and efficiency (Vuong, 2019b). Likewise, improving the quality of not only research practice but also editing and publishing is crucial to the development of Vietnamese science (Vuong, 2019b).\n\nOn the other hand, the issues of economic development, application of scientific findings, and improvement of life quality are still being questioned. Research in SSH are meant to first and foremost contribute to the fundamental understanding of one’s society – in this case, the knowledge of Vietnamese individuals about Vietnam. The process of knowledge creation, of research, also improves the professional competence of each teacher, contributing to the sustainable development of future generations. Finally, scientific research contributes directly to fostering trust in academia as well as in the education system. Research activities thus create a reasonable ecosystem to support teaching, where teachers are regularly updated with new research results, and new methods that are produced daily. The new knowledge will help teachers tailor the curriculum so students have constant access to important sources of knowledge. Besides, postgraduate education, especially programs geared towards doctoral training, also requires a student to have an in-depth understanding of scientific activities, as well as also a willingness to work hard and to emerge, themselves, in the world of academics (Vuong, 2019a). Those requirements are substantiated by publications, which in turn play an important role in creating a reputation for a university, thereby attracting students and investment. For instance, if considered separately in the context of American universities, the top 115 universities have the highest research productivity (Labaree, 2018). The quality of research is a reliable factor with the strict standards of peer review, editorial and indexing of ISI Web of Science or Scopus journals. Hence, the reputation of science-based universities is a prerequisite for forming long-term relationships with scientific investment funds. To add to it, scientific publishing entails an arduous process, even after the manuscript has been drafted, as peer-reviewing, editing and printing could take up to a year – all of which required patience and perseverance. From a perhaps more philosophical perspective, academic productivity could be considered a measure of how researchers are pushing their limits as well as the boundary of knowledge of humanity.\n\n\nData availability\n\nThe source data used in this article can be accessed from http://sshpa.com/; https://clarivate.com/webofsciencegroup/solutions/journal-citation-reports/.\n\nOpen Science Framework: F1000Research Education 4.0, https://doi.org/10.17605/OSF.IO/G6Y59 (Ho, 2020).\n\nThis project contains the following underlying data:\n\nFigure 1.csv: data presented in Figure 1\n\nFigure 2.xlsx: data presented in Figure 2\n\nFigure 3.csv: data presented in Figure 3\n\nFigure 4.csv: data presented in Figure 4\n\nFigure 5.csv: data presented in Figure 5\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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Nghi (Eds.), Reforming Higher Education in Vietnam: Challenges and Priorities. Dordrecht: Springer, Netherlands. 2010; 14–29. Publisher Full Text\n\nHo TM: F1000Research Education 4.0. 2020. http://www.doi.org/10.17605/OSF.IO/G6Y59\n\nHo TM, Do TH, Pham HH, et al.: Initial observations from Scopus data on Vietnam's international publication in the field of social sciences and humanities. Journal of Economics and Forecasting. 2017a; 12. Reference Source\n\nHo TM, Nguyen HV, Vuong TT, et al.: Exploring Vietnamese co-authorship patterns in social sciences with basic network measures of 2008-2017 Scopus data [version 1; peer review: 3 approved]. F1000Res. 2017b; 6: 1559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHo TM, Vuong TT, Vuong QH: On the sustainability of co-authoring behaviors in Vietnamese social sciences: A preliminary analysis of network data. Sustainability. 2017c; 9(11): 2142. 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Publisher Full Text\n\nVuong QH, Bui QK, La VP, et al.: Cultural evolution in Vietnam’s early 20th century: a Bayesian networks analysis of Hanoi Franco-Chinese house designs. Social Sciences & Humanities Open. 2019; 1(1): 100001. Publisher Full Text\n\nVuong QH, Ho TM, Vuong TT, et al.: Nemo Solus Satis Sapit: Trends of research collaborations in the Vietnamese social sciences, observing 2008–2017 Scopus data. Publications. 2017; 5(4): 24. Publisher Full Text\n\nVuong QH, La VP, Vuong TT, et al.: An open database of productivity in Vietnam’s social sciences and humanities for public use. Sci Data. 2018a; 5: 180188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVuong QH, Napier NK, Ho TM, et al.: Effects of work environment and collaboration on research productivity in Vietnamese social sciences: evidence from 2008 to 2017 Scopus data. Stud High Educ. 2019c; 44(12): 2132–2147. Publisher Full Text\n\nVuong TT, Nguyen H, Ho T, et al.: The (in)significance of socio-demographic factors as possible determinants of Vietnamese social scientists’ contribution-adjusted productivity: Preliminary results from 2008–2017 Scopus data. Societies. 2018c; 8(1): 3. Publisher Full Text\n\nWillinsky J: The unacknowledged convergence of open source, open access, and open science. First Monday. 2005; 10(8). Publisher Full Text\n\nWright PM, Snell SA: Toward a unifying framework for exploring fit and flexibility in strategic human resource management. Acad Manage Rev. 1998; 23(4): 756–772. Publisher Full Text"
}
|
[
{
"id": "69087",
"date": "26 Aug 2020",
"name": "John Rigby",
"expertise": [
"Reviewer Expertise Science policy",
"programme and policy evaluation and bibliometrics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reviews an increase in publication and the internationalization of publication in part of the Vietnamese science system over a recent period. Its particular focus is upon the SSHPA data. A number of policies are discussed and their effects are considered.\nThere is a very short and not very extensive description of the specific barriers to greater research productivity and internationalization of research in Vietnam.\nThe data used to reflect upon the performance of the Vietnamese science system is limited to publication data. Some inferences about policy are made on the basis of the analysis of this data, for example, on the gender of researchers, and on the issue of whether researchers are publishing for the first time. The data itself is interesting and helpful in characterizing the growth of publication and the internationalization of publication in this sector.\nThe article does not however establish many of its conclusions on the basis of the analysis. A number of the conclusions of the research are not conclusions of this research but the conclusions of other research previously published (by others, elsewhere). That discussion may be of interest but it is mainly an interrogation of and a reflection on previous research.\nA number of drafting modifications could be considered.\nThe first statement of the conclusions is close to a tautology. It could be read as 'steps to increase productivity are urgently needed to increase productivity'. If a conclusion is being offered that explains the result of policies, there is a need to restate the mechanisms/policies which have been implemented to cause the effect, in this case, to increase the productivity of and internationalization in research publication.\n\nThe immense controversy over Bayesian methods and their significance for research policy are not easy to capture in a short paragraph.\nThe conclusion on the link between research and teaching is not discussed in any detail in the literature/introduction and does not particularly follow from the analysis. It may well be a useful discussion and the conclusion may well be true as stated, but it is not clear it follows from the analysis.\nIn the Conclusion, the statement about theory and practice is not clear. Do the authors mean that policies take a long time to take effect?\nIn the abstract, it is not perhaps correct to say 'pushing competitiveness' without being more precise as to what the form of competitiveness is. It is I assume research competitiveness but there needs to be clarification.\nIn the Discussion, the following sentence is not clear: 'Recently, the subject of gender equality has gained much attention in Vietnam, especially as women continued to confirm their role in the workforce with the development of information and communication.'\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "69084",
"date": "21 Sep 2020",
"name": "Joseph Zajda",
"expertise": [
"Reviewer Expertise Comparative education research",
"qualitative methodologies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe sections to be expanded for clarification and for strengthening the argument are:\nMethods. There is a need to explain more the methodology used. The authors write that ‘The SSHPA (Social Sciences and Humanities Peer Award) database (http://sshpa.com/) was used to review the Vietnamese SSH research between 2008 and 2019’. Here they need to add that this is a part of the historical-comparative paradigm and that they use descriptive statistics (if applicable).\n\nExpand and explain more data analysis section.\n\nIn the Discussion section, elaborate more ‘The process of international cooperation in scientific research’ and ‘standards of quality’. Academic quality is one of the most important attributes here.\n\nThe Conclusion is too descriptive, rather than analytical. The authors need to stress the significance of their findings in data analysis and tables.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-152
|
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